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.

Antibodies against Platelet Glycoproteins in Clinically Suspected VITT Patients.

Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare but severe complication following COVID-19 vaccination, marked by thrombocytopenia and thrombosis. Analogous to heparin-induced thrombocytopenia (HIT), VITT shares similarities in anti-platelet factor 4 (PF4) IgG-mediated platelet activation via the FcγRIIa. To investigate the involvement of platelet-antibodies in VITT, we analyzed the presence of platelet-antibodies directed against glycoproteins (GP)IIb/IIIa, GPV and GPIb/IX in the serum of 232 clinically suspected VITT patients determined based on (suspicion of) occurrence of thrombocytopenia and/or thrombosis in relation to COVID-19 vaccination. We found that 19% of clinically suspected VITT patients tested positive for anti-platelet GPs: 39%, 32% and 86% patients tested positive for GPIIb/IIIa, GPV and GPIb/IX, respectively. No HIT-like VITT patients (with thrombocytopenia and thrombosis) tested positive for platelet-antibodies. Therefore, it seems unlikely that platelet-antibodies play a role in HIT-like anti-PF4-mediated VITT. Platelet-antibodies were predominantly associated with the occurrence of thrombocytopenia. We found no association between the type of vaccination (adenoviral vector vaccine versus mRNA vaccine) or different vaccines (ChAdOx1 nCoV-19, Ad26.COV2.S, mRNA-1273, BTN162b2) and the development of platelet-antibodies. It is essential to conduct more research on the pathophysiology of VITT, to improve diagnostic approaches and identify preventive and therapeutic strategies.

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.

Should HLA and HPA-matched platelet transfusions for patients with Glanzmann Thrombasthenia or Bernard-Soulier syndrome be standardized care? A Dutch survey and recommendations.

BACKGROUND: Glanzmann thrombasthenia (GT) and Bernard-Soulier syndrome (BSS) patients require frequent platelet transfusions and hence have an increased risk for alloimmunization against donor Human Leukocyte Antigens (HLA) when no HLA-matching is performed. Knowing that Human Platelet Antigens (HPA) are located on the platelet glycoproteins that can be absent in these patients, preventive HPA-matching may also be considered. Uniform recommendations on this topic lack in transfusion guidelines making standard practice unclear, therefore, we aimed to provide a framework for matched platelet transfusions. STUDY DESIGN AND METHODS: We conducted a targeted literature search and a national survey of Dutch (pediatric) hematologists from July to September 2021. RESULTS: We found 20 articles describing platelet transfusion policies in 483 GT-patients and 29 BSS-patients, both adults and children. Twenty surveys were returned for full analysis. All responders treated patients with platelet disorders, including GT (n = 36 reported) and BSS (n = 29 reported). Of respondents, 75% estimated the risk of antibody formation as "likely" for HLA and 65% for HPA. Formation of HLA antibodies was reported in 5 GT and in 5 BSS-patients, including one child. Fifteen respondents gave preventive HLA-matched platelets in elective setting (75%). Three respondents additionally matched for HPA in GT-patients (15%). Main argument for matched platelet transfusions was preventing alloimmunization to safeguard the effectivity of 'random' donor-platelets in acute settings. CONCLUSION: Elective HLA-matching for GT and BSS-patients is already conducted by most Dutch (pediatric) hematologists. HPA-matching is mainly applied when HPA-antibodies are formed. Based on the current literature and the survey, recommendations are proposed.

Challenging the dogma: Red blood cell-directed autoimmunity as risk factor for red blood cell alloimmunisation after blood transfusion.

Red blood cell autoimmunity and alloimmunity are potentially linked. Quantification of this association can tailor extensively matched red blood cell transfusions in patients with autoimmunity. Using an incident new-user cohort comprising 47 285 previously non-transfused, non-alloimmunised patients, we compared transfusion-induced red blood cell alloimmunisation incidences in direct antiglobulin test (DAT)-positive and control patients. Additionally, we performed case-control analyses to handle potential confounding by clinical immunomodulators. Among (IgG and/or C3d) DAT-positive patients (N = 380), cumulative red blood cell alloimmunisation incidences after 10 units transfused reached 4.5% (95% confidence interval [CI] 2.5-8.2) versus 4.2% (CI 3.9-4.5, p = 0.88) in controls. In case-control analyses, alloimmunisation relative risks among DAT-positive patients increased to 1.7 (CI 1.1-2.8). Additional adjustments for pre-DAT transfusion exposure or the extent of Rh/K mismatching did not impact results. In conclusion, while patients with DAT positivity show an intrinsically increased alloimmune red blood cell response, their absolute risk is comparable to control patients due to counteracting co-existing immunosuppressive conditions. Consequently, isolated DAT positivity in patients lacking overt haemolysis or complicated alloantibody testing does not seem to warrant extended matching strategies.

Daratumumab monotherapy in refractory warm autoimmune hemolytic anemia and cold agglutinin disease.

ABSTRACT: Autoimmune hemolytic anemia (AIHA) is a rare autoantibody-mediated disease. For steroid and/or rituximab-refractory AIHA, there is no consensus on optimal treatment. Daratumumab, a monoclonal antibody targeting CD38, could be beneficial by suppression of CD38+ plasma cells and thus autoantibody secretion. In addition, because CD38 is also expressed by activated T cells, daratumumab may also act via immunomodulatory effects. We evaluated the efficacy and safety of daratumumab monotherapy in an international retrospective study including 19 adult patients with heavily pretreated refractory AIHA. In warm AIHA (wAIHA, n = 12), overall response was 50% with a median response duration of 5.5 months (range, 2-12), including ongoing response in 2 patients after 6 and 12 months. Of 6 nonresponders, 4 had Evans syndrome. In cold AIHA (cAIHA, n = 7) overall hemoglobin (Hb) response was 57%, with ongoing response in 3 of 7 patients. One additional patient with nonanemic cAIHA was treated for severe acrocyanosis and reached a clinical acrocyanosis response as well as a Hb increase. Of 6 patients with cAIHA with acrocyanosis, 4 had improved symptoms after daratumumab treatment. In 2 patients with wAIHA treated with daratumumab, in whom we prospectively collected blood samples, we found complete CD38+ T-cell depletion after daratumumab, as well as altered T-cell subset differentiation and a severely diminished capacity for cell activation and proliferation. Reappearance of CD38+ T cells coincided with disease relapse in 1 patient. In conclusion, our data show that daratumumab therapy may be a treatment option for refractory AIHA. The observed immunomodulatory effects that may contribute to the clinical response deserve further exploration.

Generation of human antibodies targeting human platelet antigen (HPA)-1a.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS: This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS: Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION: This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

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.

Severity of haemolytic disease of the fetus and newborn in patients with a history of intrauterine transfusions in a previous pregnancy: A nationwide retrospective cohort study.

OBJECTIVE: Pregnant women who received at least one intrauterine transfusion (IUT) for haemolytic disease of the fetus and newborn (HDFN) in the preceding pregnancy are presumed to have a high likelihood of requiring IUTs again, often starting at an earlier gestational age. Our aim was to quantify these risks in a large national cohort. DESIGN: Retrospective cohort study of a nationwide Dutch database. SETTING: The Netherlands. POPULATION: All women treated in The Netherlands with IUTs for Rhesus D (RhD)- or Kell-mediated HDFN between 1999 and 2017 and their follow-up pregnancies were included. Pregnancies with an antigen-negative fetus were excluded. METHODS: Electronic patient files were searched for the number and gestational age of each IUT, and analysed using descriptive statistics and linear regression. MAIN OUTCOME MEASURES: Percentage of women requiring one or more IUTs again in the subsequent pregnancy, and gestational age at first IUT in both pregnancies. RESULTS: Of the 321 women in our study population, 21% (69) had a subsequent ongoing pregnancy at risk. IUTs were administered in 86% (59/69) of cases. In subsequent pregnancies, the median gestational age at first IUT was 3 weeks earlier (interquartile range -6.8 to 0.4) than in the preceding pregnancy. CONCLUSIONS: Our study shows that pregnant women with a history of IUTs in the previous pregnancy are highly likely to require IUTs again, and on average 3 weeks earlier. Clinicians need to be aware of these risks and ensure timely referral, and close surveillance from early pregnancy onwards. Additionally, for women with a history of IUT and their caregivers, this information is essential to enable adequate preconception counselling.

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.

Darbepoetin alfa to reduce transfusion episodes in infants with haemolytic disease of the fetus and newborn who are treated with intrauterine transfusions in the Netherlands: an open-label, single-centre, phase 2, randomised, controlled trial.

BACKGROUND: Up to 88% of infants with haemolytic disease of the fetus and newborn who are treated with intrauterine transfusions require erythrocyte transfusions after birth. We aimed to investigate the effect of darbepoetin alfa on the prevention of postnatal anaemia in infants with haemolytic disease of the fetus and newborn. METHODS: We conducted an open-label, single-centre, phase 2 randomised controlled trial to evaluate the effect of darbepoetin alfa on the number of erythrocyte transfusions in infants with haemolytic disease of the fetus and newborn. All infants who were treated with intrauterine transfusion and born at 35 weeks of gestation or later at the Leiden University Medical Center, Leiden, Netherlands, were eligible for inclusion. Included infants were randomised by computer at birth to treatment with 10 µg/kg darbepoetin alfa subcutaneously once a week for 8 weeks or standard care (1:1 allocation, in varying blocks of four and six, with no stratification). Treating physicians and parents were not masked to treatment allocation, but the research team, data manager, and statistician were masked to treatment allocation during the process of data collection. The primary outcome was the number of erythrocyte transfusion episodes per infant from birth up to 3 months of life in the modified intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT03104426) and has been completed. FINDINGS: Between Oct 31, 2017, and April 31, 2022, we recruited 76 infants, of whom 44 (58%) were randomly assigned to a treatment group (20 [45%] were allocated to receive darbepoetin alfa and 24 [55%] were allocated to receive standard care). Follow-up lasted 3 months and one infant dropped out of the trial before commencement of treatment. A significant reduction in erythrocyte transfusion episodes was identified with darbepoetin alfa treatment compared with standard care (median 1·0 [IQR 1·0-2·0] transfusion episodes vs 2·0 [1·3-3·0] transfusion episodes; p=0·0082). No adverse events were reported and no infants died during the study. INTERPRETATION: Darbepoetin alfa reduced the transfusion episodes after intrauterine transfusion treatment for haemolytic disease of the fetus and newborn. Treatment with darbepoetin alfa or other types of erythropoietin should be considered as part of the postnatal treatment of severe haemolytic disease of the fetus and newborn. FUNDING: Sanquin Blood Supply. TRANSLATION: For the Dutch translation of the abstract see Supplementary Materials section.

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.

Severe autoimmune hemolytic anemia; epidemiology, clinical management, outcomes and knowledge gaps.

Autoimmune hemolytic anemia (AIHA) is an acquired hemolytic disorder, mediated by auto-antibodies, and has a variable clinical course ranging from fully compensated low grade hemolysis to severe life-threatening cases. The rarity, heterogeneity and incomplete understanding of severe AIHA complicate the recognition and management of severe cases. In this review, we describe how severe AIHA can be defined and what is currently known of the severity and outcome of AIHA. There are no validated predictors for severe clinical course, but certain risk factors for poor outcomes (hospitalisation, transfusion need and mortality) can aid in recognizing severe cases. Some serological subtypes of AIHA (warm AIHA with complement positive DAT, mixed, atypical) are associated with lower hemoglobin levels, higher transfusion need and mortality. Currently, there is no evidence-based therapeutic approach for severe AIHA. We provide a general approach for the management of severe AIHA patients, incorporating monitoring, supportive measures and therapeutic options based on expert opinion. In cases where steroids fail, there is a lack of rapidly effective therapeutic options. In this era, numerous novel therapies are emerging for AIHA, including novel complement inhibitors, such as sutimlimab. Their potential in severe AIHA is discussed. Future research efforts are needed to gain a clearer picture of severe AIHA and develop prediction models for severe disease course. It is crucial to incorporate not only clinical characteristics but also biomarkers that are associated with pathophysiological differences and severity, to enhance the accuracy of prediction models and facilitate the selection of the optimal therapeutic approach. Future clinical trials should prioritize the inclusion of severe AIHA patients, particularly in the quest for rapidly acting novel agents.

C1-inhibitor treatment in patients with severe complement-mediated autoimmune hemolytic anemia.

Complement-mediated (CM) autoimmune hemolytic anemia (AIHA) is characterized by the destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via the lysis of donor RBCs. Because C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhances RBC transfusion efficacy. We conducted a prospective, single-center, phase 2, open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of 2 RBC units were eligible for inclusion. Four IV C1-INH doses (6000, 3000, 2000, and 1000 U) were administered with 12-hour intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation, and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and activity, peaking at 48 hours after the first dose and accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 hours. Overall, markers of hemolysis, inflammation, and complement activation remained unchanged. Five grade 3 and 1 grade 4 adverse event occurred but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent-CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

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).

Proteomic landscapes of inherited platelet disorders with different etiologies.

BACKGROUND: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. OBJECTIVES: The objective of this study was to profile the platelet proteomics signatures of IPDs. METHODS: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. RESULTS: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. CONCLUSIONS: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.

Neonatal sepsis in alloimmune hemolytic disease of the fetus and newborn: A retrospective cohort study of 260 neonates.

BACKGROUND: Among neonates with hemolytic disease of the fetus and newborn (HDFN), we aimed to describe the frequency of central-line use, indications for insertion, and incidence of confirmed and suspected sepsis, including antibiotic treatment over a 10-year surveillance period. STUDY DESIGN AND METHODS: All neonates with HDFN admitted to our neonatal intensive care unit between January 2012 and December 2021 were included in this retrospective, cohort study. Annual proportions of infants with a central-line and central-line-associated bloodstream infection (CLABSI) rates (per 1000 central-line days and per 100 infants) were evaluated. Numbers of confirmed and suspected early- and late-onset sepsis episodes were assessed over the entire study period. RESULTS: Of the 260 included infants, 25 (9.6%) were evaluated for suspected sepsis, with 16 (6.2%) having ≥1 confirmed sepsis episode. A total of 123 central-lines were placed in 98 (37.7%) neonates, with impending exchange transfusion (ET) being the most frequent indication. Of the 34 (34.7%) neonates in whom a central-line was placed due to impending ET, 11 (32.4%) received no ET. Overall CLABSI incidence was 13.58 per 1000 central-line days. Neonates with a central-line had a higher risk for confirmed late-onset infection (RR 1.11, 95% CI: 1.04-1.20) and sepsis work-up (RR 1.10, 95% CI: 1.03-1.17) compared to infants without a central-line. CONCLUSIONS: Sepsis incidence among neonates with HDFN remains high, in particular in those with a central-line. Considering the substantial proportion of neonates with a central-line without eventual ET, central-line placement should be delayed until the likelihood of ET is high.

Acute haemolytic transfusion reaction after transfusion of fresh frozen plasma in a neonate-Preventable by using solvent/detergent-treated pooled plasma?

BACKGROUND: Plasma is a commonly used blood product and is available in the form of fresh frozen plasma (FFP) or pooled solvent/detergent-treated plasma. In the Netherlands, solvent/detergent-treated plasma has become the standard product in the adult population since several years, but for neonatal use, FFP remains the product of preference. DESCRIPTION: A preterm neonate developed lung bleeding at day 8 postpartum, for which intubation and mechanical ventilation was required and transfusions with packed red blood cells and plasma, in the form of FFP, were given. Five hours after transfusion, a red discoloration of the urine occurred. An acute haemolytic transfusion was suspected, confirmed by laboratory investigations (fast decrease in haemoglobin, increased free haemoglobin, decreased haptoglobin, increased lactate dehydrogenase and a positive direct antiglobulin test [IgG 2+]). Additional research showed that the FFP product contained nonspecific auto-antibodies that reacted with the transfused erythrocytes, most test erythrocytes and the donor's own erythrocytes. CONCLUSION: A neonate experienced an acute haemolytic reaction, most probably caused by administrating a FFP product containing auto-antibodies. If transfused with solvent/detergent-treated plasma, such antibodies would have been diluted or captured. This case adds a new argument to the discussion on expanding the use of solvent/detergent-treated plasma to the paediatric population.

History and current standard of postnatal management in hemolytic disease of the fetus and newborn.

Since the discovery of the Rh blood group system in 1940, a greater understanding of hemolytic disease of the fetus and newborn (HDFN) was gained. In the years thereafter, researchers and clinicians came to the current understanding that fetal and neonatal red blood cells (RBC) are hemolyzed by maternal alloantibodies directed against RBC antigens potentially leading to severe disease. Preventative measures, such as Rhesus(D) immunoprophylaxis (RhIG), have greatly decreased the prevalence of Rh(D)-mediated HDFN, although a gap between high-income countries and middle- to low-income countries was created largely due to a lack in availability and high costs of RhIG. Other important developments in the past decades have improved the identification, monitoring, and care of pregnancies, fetuses, and neonates with HDFN. Prenatally, fetal anemia may occur and intrauterine transfusions may be needed. Postnatally, pediatricians should be aware of the (antenatally determined) risk of hemolysis in RBC alloimmunization and should provide treatment for hyperbilirubinemia in the early phase and monitor for anemia in the late phase of the disease. Through this review, we aim to provide an overview of important historic events and to provide hands-on guidelines for the delivery and postnatal management of neonates with HDFN. Secondarily, we aim to describe recent scientific findings and evidence gaps. CONCLUSION:  Multiple developments have improved the identification, monitoring, and care of pregnancies and neonates with HDFN throughout the centuries. Pediatricians should be aware of the (antenatally determined) risk of hemolysis in RBC alloimmunization and should provide treatment for hyperbilirubinemia in the early phase and monitor for late anemia in the late phase of the disease. Future studies should be set in an international setting and ultimately aim to eradicate HDFN on a global scale. WHAT IS KNOWN: • Developments have led to a greater understanding of the pathophysiology, an improved serological identification and monitoring of at-risk cases and the current pre- and postnatal treatment. WHAT IS NEW: • This review provides the pediatrician with hands-on guidelines for the delivery and postnatal management of neonates with HDFN. • Future studies should be set in an international setting with the ultimate aim of eradicating HDFN.

The interplay between GPIb/IX antibodies, platelet hepatic sequestration, and TPO levels in patients with chronic ITP.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with an incompletely understood pathophysiology but includes platelet-clearance in the spleen and liver via T cells and/or platelet autoantibodies. Strikingly, thrombopoietin (TPO) levels remain low in ITP. Platelet-glycoprotein (GP)Ibα has been described to be required for hepatic TPO generation; however, the role of GPIb antibodies in relation to platelet hepatic sequestration and TPO levels, with consideration of platelet counts, remains to be elucidated. Therefore, we examined 53 patients with chronic and nonsplenectomized ITP for whom we conducted indium-labeled autologous platelet scintigraphy and measured platelet antibodies and TPO levels. Upon stratification toward the severity of thrombocytopenia, no negative association was observed between GPIb/IX antibodies and TPO levels, suggesting that GPIb/IX antibodies do not inhibit or block TPO levels. Surprisingly, we observed a positive association between GPIb/IX antibody levels and TPO levels and GPIb/IX antibodies and platelet hepatic sequestration in patients with severe, but not mild or moderate, thrombocytopenia. In addition, platelet hepatic sequestration and TPO levels were positively associated. This collectively indicates that GPIb/IX antibodies may be associated with increased platelet hepatic sequestration and elevated TPO levels in patients with severe thrombocytopenic ITP; however, further research is warranted to elucidate the pathophysiologic mechanisms.