Disease severity in subsequent pregnancies with RhD immunization: A nationwide cohort.

BACKGROUND AND OBJECTIVES: To evaluate the severity of haemolytic disease of the foetus and newborn (HDFN) in subsequent pregnancies with RhD immunization and to identify predictive factors for severe disease. MATERIALS AND METHODS: Nationwide prospective cohort study, including all pregnant women with RhD antibodies. All women with at least two pregnancies with RhD antibodies and RhD-positive foetuses were selected. The main outcome measure was the severity of HDFN in the first and subsequent pregnancy at risk. A subgroup analysis was performed for the group of women where RhD antibodies developed after giving birth to an RhD-positive child and thus after receiving anti-D at least twice (group A) or during the first pregnancy at risk for immunization (group B). RESULTS: Sixty-two RhD immunized women with a total of 150 RhD-positive children were included. The severity of HDFN increased for the whole group significantly in the subsequent pregnancy (p < 0.001), although it remained equal or even decreased in 44% of women. When antibodies were already detected at first trimester screening in the first immunized pregnancy, after giving birth to an RhD-positive child (group A), severe HDFN in the next pregnancy was uncommon (22%). Especially when no therapy or only non-intensive phototherapy was indicated during the first immunized pregnancy (6%) or if the antibody-dependent cell-mediated cytotoxicity result remained <10%. Contrarily, women with a negative first trimester screening and RhD antibodies detected later during the first pregnancy of an RhD-positive child (group B), often before they had ever received anti-D prophylaxis, were most prone for severe disease in a subsequent pregnancy (48%). CONCLUSION: RhD-mediated HDFN in a subsequent pregnancy is generally more severe than in the first pregnancy at risk and can be estimated using moment of antibody detection and severity in the first immunized pregnancy. Women developing antibodies in their first pregnancy of an RhD-positive child are at highest risk of severe disease in the next pregnancy.

Laboratory approach for vaccine-induced thrombotic thrombocytopenia diagnosis in the Netherlands.

BACKGROUND AND OBJECTIVES: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare adverse effect characterized by thrombocytopenia and thrombosis occurring after COVID-19 vaccination. VITT pathophysiology is not fully unravelled but shows similarities to heparin-induced thrombocytopenia (HIT). HIT is characterized by the presence of antibodies against platelet factor 4 (PF4)/heparin complex, which can activate platelets in an FcγRIIa-dependent manner, whereas IgG-antibodies directed against PF4 play an important role in VITT. MATERIALS AND METHODS: We characterized all clinically suspected VITT cases in the Netherlands from a diagnostic perspective and hypothesized that patients who developed both thrombocytopenia and thrombosis display underlying mechanisms similar to those in HIT. We conducted an anti-PF4 ELISA and a functional PF4-induced platelet activation assay (PIPAA) with and without blocking the platelet-FcγRIIa and found positivity in both tests, suggesting VITT with mechanisms similar to those in VITT. RESULTS: We identified 65 patients with both thrombocytopenia and thrombosis among 275 clinically suspected VITT cases. Of these 65 patients, 14 (22%) tested positive for anti-PF4 and PF4-dependent platelet activation. The essential role of platelet-FcγRIIa in VITT with mechanisms similar to those in HIT was evident, as platelet activation was inhibited by an FcγRIIa-blocking antibody in all 14 patients. CONCLUSION: Our study shows that only a small proportion of clinically suspected VITT patients with thrombocytopenia and thrombosis have anti-PF4-inducing, FcɣRIIa-dependent platelet activation, suggesting an HIT-like pathophysiology. This leaves the possibility for the presence of another type of pathophysiology ('non-HIT like') leading to VITT. More research on pathophysiology is warranted to improve the diagnostic algorithm and to identify novel therapeutic and preventive strategies.

mRNA-1273 vaccinated inflammatory bowel disease patients receiving TNF inhibitors develop broad and robust SARS-CoV-2-specific CD8+ T cell responses.

SARS-CoV-2-specific CD8+ T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8+ T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8+ T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8+ T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8+ T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8+ T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8+ T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8+ T cells persisted and spike-specific CD8+ T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8+ T cell response.

Noninvasive fetal blood group antigen genotyping.

Noninvasive fetal blood group antigen genotyping serves as a diagnostic tool to predict the risk of hemolytic disease of the fetus and newborn in pregnancies of immunized women. In addition, fetal RHD genotyping is used as an antenatal screening to guide targeted use of immunoglobulin prophylaxis in non-immunized RhD negative, pregnant women. Based on testing of cell-free DNA extracted from maternal plasma, these noninvasive assays demonstrate high performance accuracies. Consequently, noninvasive fetal blood group antigen genotyping has become standard care in transfusion medicine.

Extensive red blood cell matching considering patient alloimmunization risk.

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) transfusions pose a risk of alloantibody development in patients. For patients with increased alloimmunization risk, extended preventive matching is advised, encompassing not only the ABO-D blood groups but also the most clinically relevant minor antigens: C, c, E, e, K, Fya, Fyb, Jka, Jkb, S and s. This study incorporates patient-specific data and the clinical consequences of mismatching into the allocation process. MATERIALS AND METHODS: We have redefined the MINimize Relative Alloimmunization Risks (MINRAR) model to include patient group preferences in selecting RBC units from a finite supply. A linear optimization approach was employed, considering both antigen immunogenicity and the clinical impact of mismatches for specific patient groups. We also explore the advantages of informing the blood bank about scheduled transfusions, allowing for a more strategic blood distribution. The model is evaluated using historical data from two Dutch hospitals, measuring shortages and minor antigen mismatches. RESULTS: The updated model, emphasizing patient group-specific considerations, achieves a similar number of mismatches as the original, yet shifts mismatches among patient groups and antigens, reducing expected alloimmunization consequences. Simultaneous matching for multiple hospitals at the distribution centre level, considering scheduled demands, led to a 30% decrease in mismatches and a 92% reduction in shortages. CONCLUSION: The reduction of expected alloimmunization consequences by incorporating patient group preferences demonstrates our strategy's effectiveness for patient health. Substantial reductions in mismatches and shortages with multi-hospital collaboration highlights the importance of sharing information in the blood supply chain.

Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs.

ABSTRACT: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti-major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S-mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

Quality assessment and harmonization of laboratories across Europe for multiple SARS-CoV-2 serology assays.

BACKGROUND AND OBJECTIVES: There is a need for conversion of SARS-CoV-2 serology data from different laboratories to a harmonized international unit. We aimed to compare the performance of multiple SARS-CoV-2 antibody serology assays among 25 laboratories across 12 European countries. MATERIALS AND METHODS: To investigate this we have distributed to all participating laboratories a panel of 15 SARS-CoV-2 plasma samples and a single batch of pooled plasma calibrated to the WHO IS 20/136 standard. RESULTS: All assays showed excellent discrimination between SARS-CoV-2 seronegative plasma samples and pre-vaccinated seropositive plasma samples but differed substantially in raw antibody titres. Titres could be harmonized to binding antibody units per millilitre by calibration in relation to a reference reagent. CONCLUSION: The standardization of antibody quantification is of paramount importance to allow interpretation and comparison of serology data reported in clinical trials in order to identify donor cohorts from whom the most effective convalescent plasma can be collected.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Materials and methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

Early, very high-titre convalescent plasma therapy in clinically vulnerable individuals with mild COVID-19 (COVIC-19): protocol for a randomised, open-label trial.

INTRODUCTION: COVID-19 convalescent plasma (CCP) is a possible treatment option for COVID-19. A comprehensive number of clinical trials on CCP efficacy have already been conducted. However, many aspects of CCP treatment still require investigations: in particular (1) Optimisation of the CCP product, (2) Identification of the patient population in need and most likely to benefit from this treatment approach, (3) Timing of administration and (4) CCP efficacy across viral variants in vivo. We aimed to test whether high-titre CCP, administered early, is efficacious in preventing hospitalisation or death in high-risk patients. METHODS AND ANALYSIS: COVIC-19 is a multicentre, randomised, open-label, adaptive superiority phase III trial comparing CCP with very high neutralising antibody titre administered within 7 days of symptom onset plus standard of care versus standard of care alone. We will enrol patients in two cohorts of vulnerable patients [(1) elderly 70+ years, or younger with comorbidities; (2) immunocompromised patients]. Up to 1020 participants will be enrolled in each cohort (at least 340 with a sample size re-estimation after reaching 102 patients). The primary endpoint is the proportion of participants with (1) Hospitalisation due to progressive COVID-19, or (2) Who died by day 28 after randomisation. Principal analysis will follow the intention-to-treat principle. ETHICS AND DISSEMINATION: Ethical approval has been granted by the University of Ulm ethics committee (#41/22) (lead ethics committee for Germany), Comité de protection des personnes Sud-Est I (CPP Sud-Est I) (#2022-A01307-36) (ethics committee for France), and ErasmusMC ethics committee (#MEC-2022-0365) (ethics committee for the Netherlands). Signed informed consent will be obtained from all included patients. The findings will be published in peer-reviewed journals and presented at relevant stakeholder conferences and meetings. TRIAL REGISTRATION: Clinical Trials.gov (NCT05271929), EudraCT (2021-006621-22).

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential.

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Children Newly Diagnosed with Fetal and Neonatal Alloimmune Thrombocytopenia: Neurodevelopmental Outcome at School Age.

OBJECTIVE: To evaluate the neurodevelopmental outcome at school age in children newly diagnosed with fetal and neonatal alloimmune thrombocytopenia (FNAIT). STUDY DESIGN: This observational cohort study included children diagnosed with FNAIT between 2002 and 2014. Children were invited for cognitive and neurological testing. Behavioral questionnaires and school performance results were obtained. A composite outcome of neurodevelopmental impairment (NDI) was used, defined, and subdivided into mild-to-moderate and severe NDI. Primary outcome was severe NDI, defined as IQ <70, cerebral palsy with Gross Motor Functioning Classification System level ≥ III, or severe visual/hearing impairment. Mild-to-moderate NDI was defined as IQ 70-85, minor neurological dysfunction or cerebral palsy with Gross Motor Functioning Classification System level ≤ II, or mild visual/hearing impairment. RESULTS: In total, 44 children were included at a median age of 12 years (range: 6-17 years). Neuroimaging at diagnosis was available in 82% (36/44) of children. High-grade intracranial hemorrhage (ICH) was detected in 14% (5/36). Severe NDI was detected in 7% (3/44); two children had high-grade ICH, and one had low-grade ICH and perinatal asphyxia. Mild-to-moderate NDI was detected in 25% (11/44); one child had high-grade ICH, and eight children were without ICH, yet for two children, neuroimaging was not performed. Adverse outcome (perinatal death or NDI) was 39% (19/49). Four children (9%) attended special needs education, three of whom had severe NDI and one had mild-to-moderate NDI. Total behavioral problems within the clinical range were reported in 12%, which is comparable with 10% in the general Dutch population. CONCLUSION: Children who are newly diagnosed with FNAIT are at increased risk for long-term neurodevelopmental problems, even those without ICH. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (Identifier: NCT04529382).

Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma.

PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A (RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M‒positive patients, compared with 84.9% for 36 RASSF1A-M‒negative patients [P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel.

The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not in antigen-experienced vaccinees.

BACKGROUND: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced FcγRIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. METHODS: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). FINDINGS: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen-experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen-experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. INTERPRETATION: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. FUNDING: LSBR1721, 1908; ZonMW10430012010021, 09150161910033, 10430012010008; DFG398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.

Outpatient convalescent plasma therapy for high-risk patients with early COVID-19: a randomized placebo-controlled trial.

OBJECTIVES: The potential benefit of convalescent plasma (CP) therapy for coronavirus disease 2019 (COVID-19) is highest when administered early after symptom onset. Our objective was to determine the effectiveness of CP therapy in improving the disease course of COVID-19 among high-risk outpatients. METHODS: A multicentre, double-blind randomized trial was conducted comparing 300 mL of CP with non-CP. Patients were ≥50 years, were symptomatic for <8 days, had confirmed RT-PCR or antigen test result for COVID-19 and had at least one risk factor for severe COVID-19. The primary endpoint was the highest score on a 5-point ordinal scale ranging from fully recovered (score = 1) or not (score = 2) on day 7, over hospital admission (score = 3), intensive care unit admission (score = 4) and death (score = 5) in the 28 days following randomization. Secondary endpoints were hospital admission, symptom duration and viral RNA excretion. RESULTS: After the enrolment of 421 patients and the transfusion in 416 patients, recruitment was discontinued when the countrywide vaccination uptake in those aged >50 years was 80%. Patients had a median age of 60 years, symptoms for 5 days, and 207 of 416 patients received CP therapy. During the 28 day follow-up, 28 patients were hospitalized and two died. The OR for an improved disease severity score with CP was 0.86 (95% credible interval, 0.59-1.22). The OR was 0.58 (95% CI, 0.33-1.02) for patients with ≤5 days of symptoms. The hazard ratio for hospital admission was 0.61 (95% CI, 0.28-1.34). No difference was found in viral RNA excretion or in the duration of symptoms. CONCLUSIONS: In patients with early COVID-19, CP therapy did not improve the 5-point disease severity score.

Postnatal treatment for children with fetal and neonatal alloimmune thrombocytopenia: a multicentre, retrospective, cohort study.

BACKGROUND: Children affected by fetal and neonatal alloimmune thrombocytopenia (FNAIT) are at risk of severe intracranial haemorrhage. Management in the postnatal period is based on sparse evidence. We aimed to describe the contemporary management and outcomes of patients with FNAIT in high-income countries. METHODS: In this multicentre, retrospective, cohort study, we set up a web-based registry for the collection of deidentified data on the management and course of neonates with FNAIT. Eight centres from seven countries (Australia, Norway, Slovenia, Spain, Sweden, the Netherlands, and the USA) participated. Eligibility criteria comprised neonates with FNAIT being liveborn between Jan 1, 2010, and Jan 1, 2020; anti-human platelet antigen (HPA) alloantibodies in maternal serum; confirmed maternal and fetal HPA incompatibility; and bleeding detected at antenatal ultrasound, neonatal thrombocytopenia (<150 × 109 platelets per L), or both in the current or previous pregnancy. Clinical data were retrieved from local medical records of the first neonatal admission and entered in the registry. The key outcome was the type of postnatal treatment given to neonates with FNAIT. Other outcomes were daily median platelet counts in the first week of life, median platelet count increment after first unmatched versus first matched transfusions, and the proportion of neonates with mild or severe bleeding. FINDINGS: 408 liveborn neonates with FNAIT were entered into the FNAIT registry, of whom 389 from Australia (n=74), Norway (n=56), Slovenia (n=19), Spain (n=55), Sweden (n=31), the Netherlands (n=138), and the USA (n=16) were included in our analyses. The median follow-up was 5 days (IQR 2-9). More neonates were male (241 [64%] of 379) than female (138 [36%]). Severe thrombocytopenia (platelet count <50 × 109 platelets per L) was reported in 283 (74%) of 380 neonates, and extreme thrombocytopenia (<10 × 109 platelets per L) was reported in 92 (24%) neonates. Postnatal platelet count nadir was higher in the no-treatment group than in all other groups. 163 (42%) of 389 neonates with FNAIT received no postnatal treatment. 207 (53%) neonates received platelet transfusions, which were either HPA-unmatched (88 [43%] of 207), HPA-matched (84 [41%]), or a combination of both (35 [17%]). The proportion of neonates who received HPA-matched platelet transfusions varied between countries, ranging from 0% (Slovenia) to 63% (35 of 56 neonates; Norway). Postnatal intravenous immunoglobulin treatment was given to 110 (28%) of 389 neonates (alone [n=19] or in combination with platelet transfusions [n=91]), with the proportion receiving it ranging from 12% (17 of 138 neonates; the Netherlands) to 63% (ten of 16 neonates; the USA) across countries. The median platelet increment was 59 × 109 platelets per L (IQR 35-94) after HPA-unmatched platelet transfusions and 98 × 109 platelets per L (67-134) after HPA-matched platelet transfusions (p<0·0001). Severe bleeding was diagnosed in 23 (6%) of 389 liveborn neonates, with one having a severe pulmonary haemorrhage and 22 having severe intracranial haemorrhages. Mild bleeding was diagnosed in 186 (48%) neonates. INTERPRETATION: Postnatal management of FNAIT varies greatly between international centres, highlighting the absence of consensus on optimal treatments. Our data suggest that HPA-matched transfusions lead to a larger median platelet count increment than HPA-unmatched transfusions, but whether HPA matching is also associated with a reduced risk of bleeding remains unknown. FUNDING: Sanquin.

Altered Fc glycosylation of anti-HLA alloantibodies in hemato-oncological patients receiving platelet transfusions.

BACKGROUND: The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%-15% of patients with chronic platelet support. Interestingly, anti-HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti-HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR-mediated phagocytosis and/or complement activation, which both are affected by the IgG-Fc glycosylation. OBJECTIVES: To characterize the Fc glycosylation profile of anti-HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome. PATIENTS/METHODS: We screened and captured anti-HLA class I antibodies (anti-HLA A2, anti-HLA A24, and anti-HLA B7) developed after platelet transfusions in hemato-oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography-mass spectrometry, we analyzed the glycosylation profiles of total and anti-HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments. RESULTS: The glycosylation profile of anti-HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients. CONCLUSIONS: These differences in composition of anti-HLA Fc-glycosylation profiles could potentially explain the variation in clinical severity between patients.

Afucosylated IgG responses in humans - structural clues to the regulation of humoral immunity.

Healthy immune responses require efficient protection without excessive inflammation. Recent discoveries on the degree of fucosylation of a human N-linked glycan at a conserved site in the immunoglobulin IgG-Fc domain might add an additional regulatory layer to adaptive humoral immunity. Specifically, afucosylation of IgG-Fc enhances the interaction of IgG with FcγRIII and thereby its activity. Although plasma IgG is generally fucosylated, afucosylated IgG is raised in responses to enveloped viruses and Plasmodium falciparum proteins expressed on infected erythrocytes, as well as during alloimmune responses. Moreover, while afucosylation can exacerbate some infectious diseases (e.g., COVID-19), it also correlates with traits of protective immunity against malaria and HIV-1. Herein we discuss the implications of IgG afucosylation for health and disease, as well as for vaccination.