BACKGROUND AND OBJECTIVES:Immunoglobulin A (IgA) deficiency is common and typically defined as <5-7 mg/dL. Individuals with absolute IgA deficiency (aIgA def) have extremely low levels, can produce allo-anti-IgA and may be at risk for anaphylactic transfusion reactions. There is no globally-accepted definition of aIgA def. We sought to determine how rare donor programmes define IgA deficiency (IgA def), how they test for it and how they manage requests for aIgA def blood products. MATERIALS AND METHODS:We queried rare blood donor programmes that participate in the International Society of Blood Transfusion Working Party on Rare Donors using an email survey sent to 39 members representing 35 programmes in 28 countries. RESULTS:Responses were obtained from 23 programmes in 16 countries. Seventeen included IgA def as a rare phenotype, with nine classifying IgA def as levels <0.05 mg/dL. Eight of the 14 programmes indicated that fulfilment of requests for IgA-deficient products requires clinical consultation and 8 programmes require patients to have detectable anti-IgA. Programmes reported provision of a small number of IgA-deficient products in the past 5 years. CONCLUSION:While respondents from rare donor programmes in 17 countries include aIgA def as a rare blood phenotype, programmes in 5 countries do not. The level of IgA used to define IgA def varies among programmes and, in some cases, across programmes within the same country. Further study is needed to better understand IgA-mediated anaphylactic transfusion reactions. A recommendation is provided for managing transfusion support of patients considered at risk.

Adaptive sampling (AS), a computational enrichment method developed for Oxford Nanopore Technologies sequencing platforms, offers a promising advance in molecular blood group diagnostics. By leveraging long-read sequencing, AS has the potential to accurately resolve complex structural variants in the RH and MNS blood group systems, while characterizing the entire blood group genome through a simple, fast and locus-adjustable protocol. As proof-of-principal, we evaluated the performance of AS using five samples with suspected complex variants in the RH and MNS systems, unresolved by standard immunohematological methods. Samples were sequenced on a PromethION P2 Solo with up to two samples per flowcell, generating 37.0-52.4 Gb of data with mean on-target coverages of 18.9-53.4x, allowing reliable variant detection. Hybrid alleles were characterized using a de novo assembly approach, whereas variants in non-recombinant regions were analyzed using both a custom in-house and the EPI2ME reference-based workflow. With reference to field-specific allele collections, 10-15% of detected alleles contained novel nonsynonymous single nucleotide variants (SNVs) or unreported exonic SNV combinations. All suspected hybrid alleles were successfully assembled and identified as GYP*401.02, RHD*03N.01, and RHD*01EL.44, representing the first fully characterized haplotypes for these variants publicly available. Overall, AS showed significant potential for advancing blood group genomics by enabling high-resolution, full-gene analysis. Its ability to support high-throughput donor genotyping and precise patient-donor matching may reduce the risk of alloimmunization and delayed hemolytic transfusion reactions, particularly in chronically transfused patients. These findings highlight AS as a powerful tool for both research and clinical applications in transfusion medicine.

Introduction:While neonates may suffer from alloimmune or secondary autoimmune diseases caused by placental transfer of maternal antibodies, primary neonatal autoimmune disease is extremely rare. Case Presentation:We present a newborn girl with fetal-onset autoimmune disease, featuring prenatal hydrothorax, progressive Coombs-positive autoimmune hemolytic anemia, and autoimmune thrombocytopenia secondary to autoantibodies against glycoprotein IIb/IIIa, which were not present in the mother. Red blood cells and platelets were repeatedly transfused during the first 5 weeks after birth. At 6 months of life, the infant displayed elevated liver enzymes and vitamin K deficiency coagulopathy due to giant cell hepatitis. There was a partial response to systemic steroids, intravenous immunoglobulins, and anti-CD20 antibodies. Complete genome analysis failed to detect a genetic cause. Conclusion:This rare case indicates that infantile Evans syndrome can already evolve during fetal development.

Background:Gestational alloimmune liver disease (GALD), previously known as neonatal hemochromatosis, is a rare maternal-fetal alloimmune disease causing severe fetal-neonatal liver failure. Understanding the immunological origin resulted in the concept of treatment with high-dose intravenous immunoglobins (IVIG) during gestation. Methods:This retrospective single-center cohort study consists of seven women with index cases of GALD, followed by a total of ten IVIG-treated singleton pregnancies. Results:Out of seven index pregnancies, five resulted in neonatal deaths. In a total of ten subsequent pregnancies all seven women at risk received antenatal IVIG (1g/kg body weight weekly) starting at median 14 weeks of gestation. Pregnancy courses were uneventful, except for one case of fetal growth restriction and preterm delivery due to severe preeclampsia. No severe maternal adverse effects of repetitive IVIG (median 23 applications) were observed. None of the infants from IVIG-treated women developed severe GALD. Two out of ten neonates displayed slightly abnormal laboratory parameters but did not require exchange transfusion or any other specific treatment. Based on standard laboratory data, we developed a scoring method to identify neonates at risk for GALD-associated liver failure. Conclusion:This case series indicates that weekly antenatal IVIG treatment is highly efficient to prevent GALD-associated neonatal liver failure. In contrast to other cases series, there were no major maternal side effects. An introduced novel laboratory scoring system to decide on early postnatal intervention requires validation in a larger number of neonates.

Background:In general, the detection of the causative drug in patients who develop allergies related to drugs is very complicated or even impossible. To date, urine and serum samples from patients who have received the drug suspected to be associated with adverse drug reactions (ex vivo antigens) have rarely been used to detect metabolite-dependent antibodies (mdab) to red blood cells (RBCs). Here, we investigated whether ex vivo drug metabolism using primary human hepatocytes might be a better alternative for obtaining the metabolites needed for the assays. Methods:Two serum samples containing mdab from 2 patients who had previously developed severe immune hemolytic anemia associated with diclofenac and 5-FU, respectively, were retested in the presence of urinary metabolites from patients and from ex vivo metabolism in primary human hepatocytes seeded in 2D monoculture. Results:Both mdab tested were detectable with urine as a source of drug metabolites, and with ex vivo drug metabolites obtained from the hepatocyte cell culture, using enzyme-treated RBCs. Conclusion:The results obtained in this study are encouraging, not only in terms of improving the detection of mdab to blood cells but also in other forms of allergies related to drugs. Ex vivo drug metabolism using hepatocyte cell culture may represent a standardized and controllable alternative to urine or serum samples for the detection of mdab to any affected human cells.

The transfusion of platelet concentrates (PC) is essential for patients with severe thrombocytopenia and life-threatening haemorrhages. The short shelf life of PC and their limited availability make it difficult to provide patients with AB0-identical and RhD-compatible PC, as recommended by the German haemotherapy guidelines. In daily practice, PC are often issued on a "first in first out" basis, but it is sometimes difficult to find compatible PCs for patients with rarer blood groups such as B or AB RhD-negative, which raises the question of which incompatibilities are acceptable and which risk minimization strategies should be applied. The PCs used in Germany are leucocyte-depleted and are prepared by the pool or apheresis method using either plasma or a mixture of plasma and a platelet additive solution (PAS). Each product has individual advantages. This article explains the immunohaematological basis of AB0 and RhD incompatibility in platelet transfusion and outlines practical strategies to minimise the risk of ABD incompatible transfusion.

In a cohort of patients treated with oral artemisinin combination therapy for uncomplicated malaria, the presence of anti-red blood cell (RBC) auto-antibodies does not correlate with the extent of post-treatment haemolysis. Patients with positive antibody screening test on d14 had higher initial parasitaemia and a higher number of once-infected RBCs throughout follow-up.

Abstract Background Artemisinin-based combination therapy (ACT) for the treatment of malaria is highly effective, well tolerated and safe. Episodes of delayed haemolysis occur in up to 57.9% of patients with severe malaria treated with intravenous artesunate, mainly caused by ‘pitting’ of infected red blood cells in the spleen and the delayed loss of these once-infected RBCs (oiRBCs). Several reports indicate that post-treatment haemolysis (PTH) also occurs in uncomplicated malaria treated with oral ACT, calling for systematic investigation. Methods A prospective observational study to identify the incidence of PTH after oral ACT, defined as increased lactate dehydrogenase activity and low haptoglobin level on Day 14 after treatment. Patients were enrolled at two study centres in Germany and Italy. Study visits took place on Days 1, 3, 7, 14 and 28. Laboratory investigations included extended clinical routine laboratory tests, quantitative PfHRP2, anti-RBC antibodies and oiRBCs. The state of semi-immunity to malaria was assessed from childhood and ongoing exposure to Plasmodium spp. as per patient history. Results A total of 134 patients with uncomplicated malaria and 3-day ACT treatment were recruited. Thirty-seven (37.4%) of 99 evaluable patients with Pf and none of 9 patients with non-Pf malaria exhibited PTH on d14. Patients with PTH had higher initial parasitaemia, higher oiRBC counts on d3 and a 10-fold decrease in oiRBCs between d7 and d14 compared with patients without PTH. In patients with PTH, loss of haemoglobin was 4-fold greater in non-Africans than in Africans (−1.3 vs −0.3 g/dl). Semi-immune African patients with PTH showed markedly increased erythropoiesis on d14 compared with not semi-immune African and non-African patients with PTH. Conclusions PTH is common in patients with uncomplicated malaria and oral ACT. While the observed loss of haemoglobin will often not be clinically relevant, it could aggravate pre-existing anaemia, warranting follow-up examinations in populations at risk.

Die medikamentös induzierte Immunhämolyse stellt eine seltene, häufig fulminant und immer wieder letal verlaufende Sonderform der autoimmunhämolytischen Anämie dar. Sie ist wenig bekannt und wird häufig übersehen. Dieser Beitrag stellt die Besonderheiten des Krankheitsbildes inkl. wesentlicher Aspekte zur Pathogenese, Klinik, Diagnostik und Therapie vor und zeigt, welche Medikamente häufig eine Immunhämolyse auslösen können.

Antibodies to red blood cells (RBCs) may hemolyze erythrocytes via Fc-mediated phagocytosis or complement-dependent. Complement activation on RBCs can be detected by C3d-direct antiglobulin test (DAT), which is the only test in immune hematology that directly targets complement. However, a positive DAT with anti-C3d cannot distinguish between C3b-mediated extravascular hemolysis, C5b-C9-mediated intravascular hemolysis and C5b-C8-mediated eryptosis. Furthermore, DAT is not suitable to estimate the strength of hemolysis. Autoimmune hemolytic anemia (AIHA) is a rare disease that is caused by autoantibodies to red blood cells that is divided in warm AIHA and in cold agglutinin disease (CAD). The causative antibodies in CAD and sometimes in warm AIHA are from the IgM class. Depending on strength of complement activation they can induce extravascular hemolysis, intravascular hemolysis and eryptosis. We studied the three types of hemolysis by use of sera from patients with CAD under various conditions. We found that additionally to the routinely applied C3d-DAT, indirect tests for complement activity (free hemoglobin and Annexin V-binding to phosphatidylserine-exposing RBCs) should be used to determine the portion of extravascular, intravascular and eryptotic hemolysis. Eryptotic hemolysis may have a significant share in clinical relevant CAD or IgM warm AIHA, which should be considered for successful treatment.

This is the first case report on two children presenting with immediate and severe hemolytic anemia following the administration of high-dose intravenous immunoglobulins (IVIGs) in the context of pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS). Hemolytic anemia was described as a significant decrease in hemoglobin and an increase in lactate dehydrogenase after the second administration of high-dose IVIGs was performed. Both patients were found to have AB blood group. One of our patients showed massive pallor, weakness, and inability to walk in association with hemolysis. However, in both cases, the anemia was self-limiting and transfusion of red blood cells was not required: both patients recovered without persistent impact. Nonetheless, we aim to draw attention to this widely unknown adverse effect of IVIG, especially in the context of PIMS-TS. We suggest determining the patient’s blood group prior to high-dose IVIG infusion and replacing the second IVIG through high-dose steroids or anticytokine therapy. Using IVIGs containing lower titers of specifically anti-A or anti-B antibodies to avoid isoagglutinin-caused hemolytic anemia is desirable; however, the information is not routinely available.

Drug induced immune hemolytic anemia (DIIHA) is rare and often associated with severe hemolytic anemia and even death. It is not well known and likely under-diagnosed. DIIHA may be caused by drug-independent autoantibodies (aab) indistinguishable from idopathic warm autoantibodies causing autoimmune hemolytic anemia, or by drug-dependent antibodies (ddab). The latter only react in the presence of the drug and can cause acute and severe intravascular hemolysis. ddab usually show a positive direct antiglobulin test (DAT) and a negative eluate. However, specialized reference laboratories are often required to perform the serological tests to confirm the diagnosis. The most common drugs to cause ddab are antibiotics (mainly piperacillin and cephalosporines), non-steroidal drugs (diclofenac) and platinum-based anticancer drugs. Stopping the drug is the first and most important approach to therapy.This paper presents the special features of the clinical picture including essential aspects of pathogenesis, clinic, diagnostics and therapy of drug induced immune hemolytic anemia.

BACKGROUND:We describe here the first patient with recurrent hemolysis related to disinfectant containing silver nanoparticles (AgNps).METHODS:A 58-year-old chemist repeatedly experienced DAT-negative (Coombs-negative) hemolysis during the last 5 years. He was treated with a number of immunosuppressive drugs including 18 times rituximab. The attempt to treat him with cyclosporine A served only to increase the rate of hemolysis. Only by chance, we revealed that the patient regularly used a hand disinfectant containing AgNps. Serological testing was performed using standard techniques. Eryptosis was measured by binding annexin to exposed phosphatidylserine (PS) of the circulating red blood cells (RBCs).RESULTS:Antiglobulin tests remained negative, and PS exposing RBCs were detected two times during the last hemolytic episodes. Hemolysis completely disappeared following discontinuation of AgNp containing products.CONCLUSION:AgNps are increasingly being used in a large variety of products. Recently, it was reported that they induce in vitro prohemolytic and procoagulant effects via oxidative stress and eryptosis. The clinical findings imply the hemolysis was provoked by the patient's regular use of cleansing products containing AgNps. Our finding might help to explain the etiology of hemolytical disorders that may remain obscure in many cases.

Background and ObjectivesThe significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry‐based erythrophagocytosis assay was established using phorbol 12‐myristate 13‐acetate (PMA)‐treated THP‐1 cells and RBCs labelled with PKH26 to assess allo‐ and autoantibodies to RBCs.Materials and MethodsTHP‐1 cells were differentiated into macrophage‐like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA‐treated THP‐1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry.ResultsWe observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti‐D and anti‐K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti‐Ch) or autoantibodies from patients and pregnant women without haemolysis.ConclusionThe flow cytometry‐based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Background McLeod syndrome (MLS) is an ultra-rare neurodegenerative X-linked disease caused by mutations in the XK gene, classified as one of the core neuroacanthocytosis syndromes. Together with the clinically very similar chorea-acanthocytosis it belongs to the heterogeneous group of ‘Huntington’s disease (HD) phenocopies’. Aims To characterize a cohort of HD phenocopies with the genetically confirmed diagnosis of McLeod syndrome. Methods This is a retrospective and prospective analysis of genotype and phenotype of sixteen McLeod cases. Results We longitudinally characterized the second largest cohort known to date. We identified novel XK mutations as well as deletions that extend into the PRRG1 gene (novel) and describe two contiguous gene deletion cases of MLS with X-linked chronic granulomatous disease (deletion also effecting the CYBB gene). This study confirms core features of MLS such as late onset hyperkinetic movements in association with neuro/myopathy, neuropsychiatric impairment, cardiac involvement, hyperCKemia. Novel aspects in this MLS series seem obstructive sleep apnea and epileptic seizure onset in childhood. Conclusions Our study expands the limited knowledge on the variable course, the various clinical manifestations and the genetic spectrum of a hereditary HD phenocopy syndrome.

ZusammenfassungEine schwere Alloimmunisierung der Schwangeren gegen erythrozytäre Blutgruppenmerkmale kann insbesondere bei Antikörpern der Spezifität Anti-K bereits vor der 20. Schwangerschaftswoche zu einer fetalen Anämie führen. Wir berichten über eine 37-jährige Schwangere (Gravida 6, Para 5) mit bekanntem Anti-K. In der Vorgeschichte kam es aufgrund einer K-Inkompatibilität in der 4. Schwangerschaft in der 34. Schwangerschaftswoche (SSW) zu einem Hydrops fetalis und in der 5. Schwangerschaft in der 22. SSW zu einem intrauterinen Fruchttod. Mittels eines nicht invasiven Pränataltests (NIPT) wurde in der aktuellen Schwangerschaft erneut ein K-positiver Fetus bestätigt. Aufgrund der Vorgeschichte erfolgte eine prophylaktische hochdosierte i. v. Immunglobulingabe (IVIG) (1 g/kgKG/Woche) ab der 14. SSW. In der 29 + 1 SSW war bei beginnender fetaler Anämie eine intrauterine Transfusion (IUT) erforderlich. Der weitere Schwangerschaftsverlauf gestaltete sich unter Fortführung der wöchentlichen IVIG-Therapie unauffällig. Das Kind kam in der 40 + 2 SSW durch Spontangeburt zur Welt. Bei positivem direktem Coombs-Test ließ sich im Eluat das mütterliche Anti-K nachweisen. Postpartal kam es bei dem Neugeborenen nur zu einem leichten Abfall der Hämoglobinkonzentration von 11,7 auf 9,5 g/dl. Eine Therapie war nicht erforderlich.

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3 , encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.

Severe alloimmunization of pregnant women against antigens on red blood cells (RBC) can lead to fetal anemia even before the 20th week of gestation, especially if anti-K antibodies are present. We report of a 37-year-old pregnant woman (G(6)P(5)) with known anti-K. She had a past history of fetal hydrops at 34th week of gestation in the 4th pregnancy and an intrauterine fetal demise at the 22nd gestational week in her 5th pregnancy caused by K-incompatibility, respectively. Using non-invasive prenatal testing (NIPT), a K-positive fetus was confirmed in the current pregnancy. In view of the previous history, prophylactic high-dose i. v. immunoglobulin (IVIG) (1 g/kg bw/week) was administered from the 14th week of gestation onwards. Due to fetal anemia, an intrauterine transfusion (IUT) was necessary at gestational week 29 + 1. The further course of the pregnancy was inconspicuous, while continuing the weekly IVIG therapy. The child was delivered at 40 + 2 weeks of gestation by vaginal delivery. The direct Coombs test was positive, and maternal anti-K could be eluted from the newborns' RBC. Post partum there was only a slight decrease in hemoglobin concentration from 11.7 g/dl to 9.5 g/dl with no therapy necessary.

BACKGROUNDDrug‐induced immune hemolytic anemia (DIIHA) is a rare but severe side effect caused by numerous drugs. Case reports and case series suggest that piperacillin‐related DIIHA may be more common among patients with cystic fibrosis (CF). However, the prevalence is speculative. The aim of this prospective, observational study was determine the prevalence of DIIHA in such affected patients.METHODS AND MATERIALSPatients with CF hospitalized for parenteral antibiotic therapy at Charité Universitätsmedizin Berlin, who had previously been exposed to IV antibiotics, were enrolled. Blood samples were collected on Days 3 and 12 of antibiotic treatment courses. Serological studies were performed using standard techniques with gel cards. Screening for drug‐dependent antibodies (ddab) was performed in the presence of the drugs and their urinary metabolites.RESULTSA total of 52 parenteral antibiotic cycles in 43 patients were investigated. Ddab against piperacillin were detected in two patients (4.7%). The direct AHG was positive with anti‐IgG only in both patients. However only one of these patients developed mild immune hemolytic anemia. Both patients had been repeatedly treated with piperacillin without any evident hemolysis. There was no correlation between the exposure to piperacillin and the prevalence of ddab.CONCLUSIONOur prospective study indicates that piperacillin‐induced ddab occur more frequently in patients with CF than previously suggested. The question related to the significance of piperacillin‐dependent antibodies may reflect new aspects in this field.