Extracorporeal photopheresis induces NETosis in neutrophils derived from patients with chronic graft-vs-host disease.

INTRODUCTION: Extracorporeal photopheresis (ECP) is considered an effective treatment for patients with chronic graft vs host disease (cGVHD) and demonstrates efficacy in ameliorating GVHD. The mechanism by which ECP acts against cGVHD is not fully understood. Preliminary observations have hinted at the potential involvement of neutrophil extracellular traps (NETs) formation in the pathogenesis of cGVHD. We aimed to assess the influence of ECP on the formation of NETs in patients with cGVHD as a potential mechanism in this setting. METHODS: Patients treated with ECP for cGVHD at the Rabin Medical Center were included in this study. Blood samples were obtained at three different time points: before starting an ECP cycle, at the end of the first day of treatment, and 24 h following the initiation of the ECP treatment cycle. Neutrophils were harvested from all blood samples. NET formation was assessed by measurement of NET-bound specific neutrophil elastase activity and by immunofluorescence staining. RESULTS: Six patients (two females and four males) with cGVHD were included in the study. We observed a significant increase in NET formation among all six patients following ECP. Net-bound specific neutrophil elastase activity was elevated from a median value of 2.23 mU/mL (interquartile range [IQR] 2.06-2.47 mU/mL) at baseline to a median value of 13.06 mU/mL (IQR 10.27-15.97 mU/mL) immediately after the treatment and to a peak median value of 14.73 mU/mL (IQR 9.6-22.38 mU/mL) 24 h following the initiation of the ECP cycle. A qualitative assessment of NET formation using immunofluorescence staining has demonstrated markedly increased expression of citrullinated histone H3, a marker of NET formation, following ECP treatment. CONCLUSIONS: Our preliminary data indicate that ECP induces NET formation among patients with cGVHD. The contribution of increased NET formation to the therapeutic effect of cGVHD should be further investigated.

Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity.

Introduction: The success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. Methods: We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls. Results: In contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. Discussion: These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.

Reciprocal interactions between innate immune cells and astrocytes facilitate neuroinflammation and brain metastasis via lipocalin-2.

Brain metastasis still encompass very grim prognosis and therefore understanding the underlying mechanisms is an urgent need toward developing better therapeutic strategies. We uncover the intricate interactions between recruited innate immune cells and resident astrocytes in the brain metastatic niche that facilitate metastasis of melanoma and breast cancer. We show that granulocyte-derived lipocalin-2 (LCN2) induces inflammatory activation of astrocytes, leading to myeloid cell recruitment to the brain. LCN2 is central to inducing neuroinflammation as its genetic targeting or bone-marrow transplantation from LCN2-/- mice was sufficient to attenuate neuroinflammation and inhibit brain metastasis. Moreover, high LCN2 levels in patient blood and brain metastases in multiple cancer types were strongly associated with disease progression and poor survival. Our findings uncover a previously unknown mechanism, establishing a central role for the reciprocal interactions between granulocytes and astrocytes in promoting brain metastasis and implicate LCN2 as a prognostic marker and potential therapeutic target.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Generation of vascular chimerism within donor organs.

Whole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

An Early Unexpected Immune Thrombotic Thrombocytopenic Purpura Relapse Associated with SARS-CoV-2 Infection: A Case Report and Literature Review.

SARS-CoV-2 has been reported as a possible triggering factor for the development of several autoimmune diseases and inflammatory dysregulation. Here, we present a case report of a woman with a history of systemic lupus erythematosus and antiphospholipid syndrome, presenting with concurrent COVID-19 infection and immune thrombotic thrombocytopenic purpura (TTP). The patient was treated with plasma exchange, steroids, and caplacizumab with initial good response to therapy. The course of both TTP and COVID-19 disease was mild. However, after ADAMTS-13 activity was normalized, the patient experienced an early unexpected TTP relapse manifested by intravascular hemolysis with stable platelet counts requiring further treatment. Only 3 cases of COVID-19 associated TTP were reported in the literature thus far. We summarize the literature and suggest that COVID-19 could act as a trigger for TTP, with good outcomes if recognized and treated early.

Willingness and concerns of transfusion-dependent hematological patients toward the option of home transfusion therapy.

BACKGROUND: One of the main obstacles of providing home-based palliative care to transfusion-dependent hematology patients is the lack of home transfusions services. While healthcare professionals are concerned with safety and cost of home transfusions, the attitude of the patients toward home transfusions are mostly unknown. AIM: To obtain quantitative data regarding the willingness and concerns of transfusion-dependent patients with hematological diseases toward the option of home transfusions. DESIGN: A cross sectional survey including a self-administered questionnaire in one of the three main spoken languages in Israel was administered to patients in 17 hospital hematology outpatient clinics between May 2019 and March 2020. RESULTS: About 52% of 385 patients that participated in the survey preferred home transfusions to hospital transfusions. Gender, age, education, or type of disease were not associated with preference for home transfusions, nor were hospital location or its size. The likelihood to prefer home transfusions was significantly higher among the Hebrew-speakers and those who had not experienced adverse effects previously. The most significant factor associated with preference of home transfusions was a perceived negative effect of hospital-based transfusion on quality of life. The main reason to reject home transfusions was fear of possible adverse effects and concerns over losing contact with the medical staff at the treating hospital. CONCLUSION: These data suggest that a significant portion of transfusion-dependent patients in Israel view home transfusions as a preferred treatment option and that its successful implementation requires maintaining ongoing contact with the treating hospital.

Long-term outcomes of catheter-based intervention for clinically significant paravalvular leak.

BACKGROUND: In most centres, clinically significant percutaneous paravalvular leak (PVL) closure following valve replacement surgery is reserved for those considered high-risk for surgery. There is a paucity of data regarding the long-term outcomes of these patients. AIMS: Our goals were to assess the long-term outcomes of patients undergoing percutaneous PVL closure. METHODS: A total of 100 consecutive transcatheter PVL closure procedures (74 mitral, 26 aortic) were performed in 95 patients between February 2005 and August 2019 at our hospital. Data collected included procedural success rates, indication-specific outcomes and mortality. RESULTS: Mean follow-up was 5.6±6.1 years, mean age 62.6±15.2 years, and 45.4% were female. The device was successfully implanted in 88 procedures (88.0%). Patients who presented with heart failure (n=57) had a significant improvement in NYHA classification (29.2% Class III/IV versus 100.0%, p<0.001). For patients who presented with haemolytic anaemia (n=38), haemoglobin increased (11.94±1.634 vs 9.72±1.49, p<0.001) and LDH levels were reduced (1,354.90±1,225.55 vs 2,039.40±1,347.20, p<0.001) following the procedure. Rates of mortality were 3.8% at 90 days, 15.6% after 1 year, and 27.2% after 5 years. CONCLUSIONS: For patients who are deemed intermediate- to high-risk for repeat surgery, transcatheter PVL closure shows reasonable clinical success rates, with a significant improvement in symptoms, and a relatively low rate of periprocedural complications.

[COVID-19 - BLOOD GROUPS, ANTIBODIES AND CONVALESCENT PLASMA].

INTRODUCTION: Israel is currently struggling with the Coronavirus Disease (COVID-19) caused by SARS CoV-2. Transmission is increasing, with higher morbidity and mortality among populations at risk. Over-representation of blood type A was reported in COVID-19 patients with increased respiratory failure, while blood type O seems to have a protective effect. This may be caused by interference of anti-A antibodies in viral binding to the ACE receptor, different neutralization antibodies potency or variations in the stability of von Willebrand factor (VWF) multimers in different blood types. Since transfusion of convalescent COVID19 Plasma (CCP) is an accepted therapeutic modality, the Ministry of Health initiated a national project whereby CCP is collected by Magen David Adom (MDA) Blood Services using apheresis procedures and transfusions are approved by an experts committee, as part of the clinical trial or as compassionate treatment. Preliminary analysis of 49/170 patients treated so far shows improvement in 49%, with important relations with the anti-SARS-CoV-2 IgG antibodies level in the transfused plasma. Anti-SARS-CoV-2 antibodies were found in 83% of 1100 CCP donors, but a 13% decrease in antibodies level was detected in repeat donations. Blood type A was more predominant among CCP donors, when compared to MDA blood donors' data. A transfusion of CCP is a feasible and relatively safe therapeutic modality, mainly for patients with moderate COVID 19. CCP also serves as a source for the production of hyperimmune globulin for the treatment of COVID 19 and for passive immunization for populations at risk.

Red blood cell alloimmunization prevalence and hemolytic disease of the fetus and newborn in Israel: A retrospective study.

Hemolytic disease of the fetus and newborn (HDFN) is a severe form of anemia caused by maternal antibodies against fetal red blood cells (RBC) that can cause intrauterine and perinatal morbidity and mortality. The prevalence and specificities of alloantibodies among Israeli pregnant women and clinical outcomes for their fetuses and newborns are unknown. STUDY DESIGN AND METHODS: A retrospective study of women who gave birth between January 1, 2011, and December 31, 2011, was performed. Data were obtained for obstetric admissions from 16 of 27 hospitals, which included results of maternal ABO, D, antibody screens, antibody identification, and requirements for intrauterine or newborn exchange transfusions. RESULTS: Data on 90 948 women representing 70% of all births during 2011 were analyzed. Antibody screen was positive in 5245 (5.8%) women. Alloantibodies, excluding anti-D titer (<16) were identified in 900 (1.0%) women. Of 191 D- women, 75 (39.3%) had anti-D titer of 16 or greater. Other common clinically significant antibodies were anti-E (204, 23%), anti-K (145, 16%), and anti-c (97, 10.8%) alone or in antibody combinations. Multiple alloantibodies were observed in 132 of 900 (15%) of women. Severe HDFN developed in 6.8% (9/132) of these pregnancies. Seventeen fetuses and newborns (0.02% of births) including one set of twins required RBC transfusions. Two fetuses whose mothers had multiple alloantibodies received intrauterine transfusions; one of them was hydropic and died. CONCLUSION: The prevalence of RBC alloantibodies was 1.0% among Israeli pregnant women. Transfusion was required in 0.02% of the fetuses and newborns. Severe HDFN developed in 6.8% of pregnancies with multiple maternal alloantibodies.

Disruption of the tumour-associated EMP3 enhances erythroid proliferation and causes the MAM-negative phenotype.

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.

Alloimmunization and autoimmunization in adult transfusion-dependent thalassemia patients: a report from a comprehensive center in Israel.

Patients with beta thalassemia major (TM) are transfusion-dependent (TD) since early childhood and for life. Development of alloantibodies and autoantibodies against red blood cell (RBC) antigens is increasingly recognized as a significant transfusion hazard, especially among heavily transfused patients. The aim of this study is to assess RBC alloimmunization and autoimmunization rates in TD TM patients treated in our Comprehensive Center of Adult Thalassemia, Hemoglobinopathies and Rare Anemias. TD TM patients, regularly transfused every 2-3 weeks, were included in the study. Clinical and RBC transfusion records, including RBC antibodies, since diagnosis in early childhood, were retrieved from patients' files and from the blood bank database. Forty TD TM patients, > 18 years of age, were included in the study. Alloimmunization was demonstrated in 17 (42.5%) patients. Thirty-four alloantibodies were detected, with the most frequent being RH related (12 of 34, 35.3%) followed by those of the Kell system (8 of 34, 23.5%). Age at first transfusion was positively related to the probability of developing alloantibodies (p = 0.02). Splenectomy was found to be correlated with developing alloantibodies (p = 0.016). Logistic regression analysis of the lifelong probability of developing alloantibodies on the age at first transfusion and splenectomy demonstrates a strong positive relationship (p = 0.002). A substantially high rate of alloimmunization was found among adult TD TM patients. Early initiation of RBC transfusions, avoidance of splenectomy and extended Rh and K antigen matching, can reduce the incidence of alloimmunization in TD TM patients.

Prothrombin Complex Concentrate before Urgent Surgery in Patients Treated with Rivaroxaban and Apixaban.

INTRODUCTION: Patients treated with direct Xa inhibitors may require urgent surgery. Administration of prothrombin complex concentrate (PCC) in this setting is common; however, it is based on limited experience in healthy volunteers. OBJECTIVE: To characterize the population receiving PCC for apixaban/rivaroxaban reversal prior to an urgent surgery and evaluate its efficacy and safety. METHODS: This was a retrospective study in 2 tertiary hospitals. Bleeding was evaluated based on surgical reports, hemoglobin drop, and the use of blood products or additional PCC during 48 h. Safety measures were thrombotic complications and 30-day mortality. RESULTS: Sixty-two patients aged 80.7 ± 9 years, treated with apixaban (39.63%) or rivaroxaban (23.37%), received PCC before an urgent surgery/procedure. Most underwent abdominal operation (61%), orthopedic surgery (13%), or transhepatic cholecystostomy insertion (10%). Bleeding during surgery was reported in 3 patients (5%), no patient required additional PCC, and 16 patients (26%) received packed cells (median: 1 unit, range: 1-5). The 30-day mortality and thrombosis rates were 21% (n = 13) and 3% (n = 2), respectively. The cause of death was related to the primary disease, most commonly sepsis. No patient died due to bleeding/thrombosis. CONCLUSIONS: Our results support the use of PCC to achieve hemostasis in patients treated with Xa inhibitors prior to an urgent surgery.

Extended Follow-up of an Educational Intervention Encouraging Appropriate Use of Blood Transfusions.

INTRODUCTION: A restrictive transfusion strategy of packed red blood cells (PRBCs) has been associated with at least non-inferior patient outcomes in a variety of clinical settings. In December 2014, we conducted an educational intervention which consisted of an oral presentation and computerized notifications at a single tertiary medical center. OBJECTIVE: The aim of this study was to examine the long-term effects of a simple and low-cost educational intervention aimed to promote awareness to transfusion guidelines. METHODS: We retrospectively analyzed all PRBC transfusions ordered between 2014 and 2017. The primary end point was defined as the percentage of PRBC transfused to patients with hemoglobin (Hb) ≥8 g/dL. RESULTS: Between 2014 and 2017, a total of 27,475 PRBCs were transfused in our medical center. There was a continuous reduction in the percentage of PRBCs transfused at a Hb level ≥8 g/dL between 2014 and 2017, with a matching increase in the PRBC percentage trans-fused at Hb <7 g/dL (OR reduction of 42%, 95% CI 0.54-0.62 and OR increase of 68% [95% CI 1.56-1.81], respec-tively). CONCLUSION: A simple educational intervention likely contributed to sustained improvement in the appropriateness of PRBC transfusions.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology: Report of the Dubai, Copenhagen and Toronto meetings.

BACKGROUND AND OBJECTIVES: The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session. METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented. RESULTS AND CONCLUSIONS: Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies.