SARS-CoV-2 infection triggers a strong antibody response toward nucleocapsid protein (NP), suggesting its extracellular presence beyond intravirion RNA binding. Our co-culture experiments show NP decorates infected and proximal uninfected cell surfaces. We propose a mechanism whereby extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated glycosaminoglycans using its RNA-binding sites, facilitated by the flexible, positively charged linker. Coating uninfected lung-derived cells with NP attracted anti-NP IgG from lung fluids and sera of COVID-19 patients. Immune recognition was significantly higher in moderate versus mild COVID-19. Binding of anti-NP IgG in sera generated clusters, triggering C3b deposition via the classical complement pathway on SARS-CoV-2 non-susceptible cells co-cultured with infected cells. The heparin analog enoxaparin outcompeted NP binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings reveal how extracellular NP may exacerbate COVID-19 damage and suggest preventative therapy avenues.

INTRODUCTION:Hyperhemolytic syndrome (HHS) is a severe form of delayed transfusion reaction primarily described in sickle cell anemia patients which is characterized by a hemoglobin decrease to pre-transfusion levels or lower, often with reticulocytopenia and no evidence of auto- or allo-antibodies.CASE PRESENTATION:We present two cases of severe HHS in patients without sickle cell anemia refractory to treatment with steroids, immunoglobulins, and rituximab. In one case, temporary relief was achieved with eculizumab. In both cases, plasma exchange resulted in a profound and immediate response allowing for splenectomy and resolution of hemolysis.DISCUSSION/CONCLUSION:We discuss the pathophysiology of HHS, its presentation and treatment and expand on the possible role of plasma exchange in this setting.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable “sterilizing immunity” at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Red blood cell (RBC) adhesion to vascular endothelial cells (EC) is considered a potent effector of circulatory disorders, and its enhancement is implicated in the pathophysiology of numerous conditions, mainly hemoglobinopathies. The actual RBC/EC interaction is determined by both cellular and plasmatic factors, and the differentiation between them is essential for understanding its physiological implications. Yet, RBC/EC adhesion has been studied predominantly in protein-free media. To explore the plasma contribution to RBC/EC adhesion, we examined the adhesion of human RBC to human vascular endothelial cells in the presence of fresh frozen plasma (FFP) and compared it to that in a protein-free phosphate-buffered saline (PBS).RBC from blood samples freshly-collected from five healthy donors and from fifteen units of packed RBC units were used. The same FFP sample was used in all measurements.In FFP, the RBC form strongly adherent aggregates, which are dispersed as the shear stress (τ) increases to 3.0 Pa, and even at 5.0 Pa a large portion of the RBC are still adherent. In PBS, the RBC are singly dispersed and their adhesion becomes insignificant already at τ = 0.5 Pa. No cross-correlation was found between the adhesion in PBS vs. that in FFP at the same τ. However, in both media, under conditions that form singly dispersed adherent RBC, an inverse correlation between RBC/EC adhesion in PBS vs. that in FFP was observed.This study clearly implies that for understanding the physiological relevance of RBC/EC adhesion it should be determined in plasma.

Anti-RhD antibodies are widely used in clinical practice to prevent immunization against RhD, principally in hemolytic disease of the fetus and newborn. Intriguingly, this disease is induced by production of the very same antibodies when an RhD negative woman is pregnant with an RhD positive fetus. Despite over five decades of use, the mechanism of this treatment is, surprisingly, still unclear. Here we show that anti-RhD antibodies induce human natural killer (NK) cell degranulation. Mechanistically, we demonstrate that NK cell degranulation is mediated by binding of the Fc segment of anti-RhD antibodies to CD16, the main Fcγ receptor expressed on NK cells. We found that this CD16 activation is dependent upon glycosylation of the anti-RhD antibodies. Furthermore, we show that anti-RhD antibodies induce NK cell degranulation in vivo in patients who receive this treatment prophylactically. Finally, we demonstrate that the anti-RhD drug KamRho enhances the killing of dendritic cells. We suggest that this killing leads to reduced activation of adaptive immunity and may therefore affect the production of anti-RhD antibodies.

Background: Donated blood is stored in the blood bank as packed red blood cell units. In the process of packed cells preparation, the red blood cells (RBCs) are subjectedto high level of shear stress, which can induce alterations in their properties. In the present study, we examined the effect of packed RBCs preparation (which included leuko-filtration) on red cell deformability. Methods: Blood samples were collected from 25 healthy donors and from corresponding units of packed RBCs. The portion of undeformable cells (%UDFC) was determined for each sample. Results: The median value of %UDFC was equal to 6.75 +/- 0.70 %, for freshly-donated RBCs, and to 6.36 +/- 0.51 %, for packed cells. Wherein, %UDFC may increase or decrease following packed cells preparation, depending upon the initial portion of undeformable cells. Conclusion: Likely, exposure of RBCs to high shear stress, during packed cells preparation, induces opposing effects: (a) removal/destruction of rigid (undeformable) cells, thereby reducing their total amount (i.e., decreasing the %UDFC) on the one hand, and (b) mechanical damage to the cell membrane and subsequent reduction of the cell deformability (thereby increasing the %UDFC) on the other. As a consequence, the final impact of packed cells preparation is primarily determined by the initial state of erythrocytes in the blood of the donor.

BackgroundHemolytic 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 MethodsA 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.ResultsData 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.ConclusionThe 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.

Objective This study aimed to examine the donor-to-donor variability in the deformability of red blood cells (RBCs) from freshly collected blood donations (F-RBC) and packed RBCs. Background Packed RBCs are supplied for transfusion by the first-in-first-out (FIFO) criterion, assuming that their quality is the same for packed RBCs with equal storage duration. To challenge this notion, we determined the deformability of F-RBC and packed RBCs stored for different durations. Methods Three RBC groups were employed: A. 79 samples of F-RBC; B. 76 samples of packed RBC units, randomly used for transfusion at different storage durations; and C. 65 samples of outdated packed RBCs stored for 35 to 37 days. All packed RBC units were non-leukofiltrated and stored in Citrate-phosphate-dextrose solution with adenine (CPDA-1). RBC deformability was determined using a computerised cell-flow properties analyser, which monitors the shape change of cells directly visualised in a narrow-gap flow chamber and provides the cells' deformability distribution in a large RBC population. Results The F-RBC deformability exhibited a wide-range inter-donor variability. The cold storage of packed RBCs exerted a mild reduction of deformability, which became significant, compared to the initial inter-donor variability, only after 3 weeks of storage. Conclusion Packed RBCs are generally supplied for transfusion by the FIFO criterion based on the assumption that the storage duration is a key factor of RBC quality. This study demonstrates that the deformability of red blood cells is significantly different in donors, and substantial variability persists throughout the entire process of their storage. Therefore, the FIFO criterion is not sufficient for assessing the RBC deformability, which should, therefore, be specifically characterised for each unit.

Background: We determined the temporal pattern of early SARS-CoV-2 IgG response in patients with mild COVID-19, and sought to identify predictive clinical and laboratory features. Methods: Serum samples were prospectively obtained from 111 convalescent COVID-19 patients, staying in dedicated Isolation–hotels, and tested for the presence of SARS-CoV-2 IgG by anti-S1 protein ELISA. Results: SARS-CoV-2 IgG was detected in 78 (70.3%) patients tested within the first month from diagnosis. While highly variable between patients, the rate of antibody detection generally increased with time, from 47.1% to 93.8% at the first and fourth weeks from diagnosis, respectively, with the largest shift observed between the second and third week. Notably, the presence of more profound symptoms at presentation, namely, fever and chills, positively and independently correlated with early antibody response. IgG-positive patients had higher ferritin levels (p=0.039). Older age (p<0.001) and increased CRP levels (p=0.001) were associated with higher SARS-CoV-2 IgG levels. Conclusions: The identified temporal pattern along with the correlation between inflammation-related clinical and laboratory parameters and early IgG response in patients with mild COVID-19, could provide a basis for better prediction and understating of the immune response to SARS-CoV-2, and inform therapeutic donor-plasma selection.

Autoimmune cytopenias (AIC) following allogeneic hematopoietic stem cell transplantation (HSCT) may cause significant morbidity and mortality and are often challenging to treat. We present a case of a pediatric patient with primary myelofibrosis of infancy caused by VPS45 protein deficiency, who developed severe refractory hemolytic anemia and immune-mediated thrombocytopenia 3.5 months following HSCT. After the failure of several treatments, he received daratumumab, an anti-CD38 specific antibody, and demonstrated fast and sustained response. The only side effect was delayed recovery of humoral immunity. Daratumumab, by targeting antibody-producing plasma cells, may be a valid treatment option for refractory post-HSCT AIC.

Under the current regulations, blood units, processed as packed red blood cells (PRBC), are stored in the cold room, where the temperature should be kept between 2°C and 6°C, presumably to minimize the chances of contamination and the storage-induced cell lesion. However, noting the water anomaly, we have questioned whether this temperature range indeed provides the optimal conditions for minimizing storage lesion. Differing from other fluids, water is at its' most compact packing at 4°C, below which it undergoes numerous abnormal structural processes, among them the breakage of hydrogen bonds, which affects the water structure and interactions with neighboring molecules. Concomitantly, the water density reaches a maximal value at 4°C, implying that during the routine cold storage PRBC are subjected to repeated temperature fluctuations below and above the 4°C critical temperature, and the respective volume/density variations. As these anomalies are expected to induce instability of membrane-associated water1, 2 and cytoskeleton organization,3 we hypothesized that the current routine cold storage impairs the cell function. RBC are characterized by their deformability, that is, the cells' ability to change their shape under flow-induced shear stress. This is essential for adequate flow, especially in the microcirculation,4 and plays an important role in splenic RBC sequestration. In addition to its own physiological role, RBC deformability can be used for general characterization of the RBC state,5 as it is an integrated characteristic of the cell's features. In recent studies, we have demonstrated that the deformability of transfused PRBC plays a key role in transfusion outcome, as expressed by the transfusion-induced change in the recipients' hemoglobin level6 and blood flow4; both improved with increasing PRBC deformability. On these grounds, we used the PRBC deformability to test the hypothesis that the current routine of storing PRBC at 2°C-6°C is not optimal. It has been previously shown, by us,4, 6 that the routine storage of blood units at 2°C-6°C is associated with a reduction of RBC deformability, which becomes significant around 3 weeks of storage. In accordance with that, in the present study, the deformability of group-1 PRBC (stored at 2.5°C-5.5°C) decreased considerably from day 1 to day 35, whereas the change in the deformability of group-2 PRBC (stored at 5°C-6°C) was insignificant (Table 1). As noted above, of particular relevance to transfusion outcome is the fraction of the low deformable cell in the transfused PRBC population, as this fraction was found in our previous study to inversely correlate with the transfusion-induced increment of the recipients' hemoglobin.6 Table 1 shows that during 35 days of storage, the %LDFC in group-1 increased considerably (from 36.6% to 50.8%, P = 0.0022), while in group 2 PRBC it remained unchanged (36.6%-39.3%, P = 0.2). The effect of storage temperature is especially well demonstrated when comparing the deformability of the individual PRBC units in the two groups. As shown in Supporting Information Figure S1A, for each unit the RBC deformability, expressed by MER (Supporting Information Figure S1A), was higher when stored under 5°C-6°C (group-2) than under the current routine conditions (2.5°C-5.5°C, group-1). This is further demonstrated in Supporting Information Figure S1B, showing that for each PRBC unit the %LDFC in the PRBC population was lower when stored under 5°C-6°C than under 2.5°C-5.5°C. This study shows that storing PRBC under conditions that expose them to the temperature fluctuation around 4°C (group-1), induces a considerable decrease in the RBC deformability, whereas when the exposure to these conditions are avoided (group-2), the RBC deformability is unchanged during storage. As noted above, RBC deformability is an integrative property, and its impairment expresses, in particular, changes in the cell membrane and cytoskeleton structure. Therefore, the finding that storage under group-2 conditions (5°C-6°C) is not associated with damage to RBC deformability may imply that the storage-induced lesion would be smaller when the PRBC are spared from the temperature fluctuations below and above 4°C. The current conventional storage duration has been determined according to the Food and Drug Administration (FDA) requirement for 75% survival of transfused PRBC in the recipient's blood 24 h posttransfusion. On these grounds, the storage-induced change in the percent of a low-deformable cell (%LDFC, see details in Supporting Information), which was considerable in group-1 but insignificant in group-2, is of particular interest. Previous studies have suggested that RBC with low deformability are prone to accelerated clearance from the vascular system.6 In accordance with that, we have found that the fraction of the relatively rigid RBC, as expressed by %LDFC, in the population of transfused PRBC, inversely correlates with the Hb increment in the recipients' blood, measured right after completion of the transfusion. This implies that this measure reflects the survival of transfused PRBC in the recipients' vascular system. Therefore, the fact that the portion of the relatively rigid RBC in group-2 is unchanged during storage may suggest that storing PRBC under conditions that avoid temperature fluctuations below/above 4°C would increase their survival rate and possibly enable longer storage duration. In conclusion, the present study, although of limited scope, demonstrates that the PRBC “quality”, as expressed by the cell deformability, is maintained better when PRBC are not exposed to repeated temperature fluctuations below/above the 4°C critical temperature, where the cells are subjected to the water anomalies. To substantiate this conclusion, further comprehensive study of this phenomenon is needed, but in this experimental study, it is proposed to review the long-standing storage order that is currently practiced in the blood bank. All authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A 25-year-old male was admitted for evaluation of fatigue, and was found to have severe anaemia with a haemoglobin level of 6.4 gr%, MCV 118 fL, 116 000 reticulocytes/μL (7%), 1800×109/L neutrophils and thrombocytopaenia of 37×109/L. Biochemical parameters were compatible with haemolysis as elevated LDH (1314 U/L) and low haptoglobin (2 mg/dL). Bone marrow biopsy revealed hypocellular bone marrow (15%), leading to a diagnosis of aplastic anaemia dominated by a paroxysmal nocturnal haemoglobinuria (PNH) clone in 60% of neutrophils. As no matched donors were identified for allogeneic stem cell transplantation (SCT), the patient was vaccinated with a conjugated vaccine against Neisseria meningitidis (Nimenrix, GSK), designed to prevent invasive meningococcal disease (IMD) by serogroups A,C,W135 and Y, followed 2 weeks later by initiation of PNH treatment with the complement inhibitor eculizumab (Soliris, Alexion). Four days after the first eculizumab dose, the patient was admitted with a 40°C fever and rigors in the absence of nuchal rigidity or evidence of focal infection, and treatment with empirical intravenous ceftriaxone and ampicillin was started. Twenty-four hours later, Neisseria meningitidis serogroup Y growth was noted in blood cultures. The patient completed 5 days of intravenous ceftriaxone, followed by a 7-day treatment with oral ciprofloxacin. The patient did not have any risk factors for meningococcal infection but was living with his parents and 12 siblings, all were screened for meningococcal pharyngeal carriage and found to be negative. To date, the patient has been treated with repeated blood transfusions and has declined allogeneic SCT. Eculizumab treatment has not yet been renewed. Neisseria meningitidis is a cause of bacterial meningitis, sepsis, pneumonia, and localized infections. Approximately 10% of the population feature pharyngeal Neisseria meningitidis carrier state. Populations at risk for invasive meningococcal diseases (IMD) include splenectomized patients, crowded household contacts, smokers, and individuals suffering from complement deficiency or antecedent viral infection. Eculizumab, a humanized monoclonal antibody used for treatment of PNH, has been reported to predispose to infection with encapsulated bacterial strains including Neisseria meningitidis. Thus, meningococcal vaccination has been recommended at least 2 weeks prior to initiation of eculizumab treatment [[1]Hillmen P. Muus P. Röth A. Elebute M.O. Risitano A.M. Schrezenmeier H. et al.Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal hemoglobinuria.Br J Haematol. 2013; 162: 62-73Crossref PubMed Scopus (255) Google Scholar], but because of enhanced haemolysis observed in patients already treated with eculizumab and then vaccinated against serogroup B meningococcal infection, it is recommended to vaccinate this group of patients within a week of eculizumab infusion. Nevertheless, 63 cases of IMD following prior vaccination were reported by the manufacturer, including eight cases fully described in the medical literature [2Vicente D. Esnal O. Pérez-Trallero E. Fatal Neisseria meningitidis serogroup X sepsis in immunocompromised patients in Spain. Virulence of clinical isolates.J Infect. 2012 Feb; 64: 184-187Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 3Struijk G.H. Bouts A.H. Rijkers G.T. Kuin E.A. ten Berge I.J. Bemelman F.J. Meningococcal sepsis complicating eculizumab treatment despite prior vaccination.Am J Transplant. 2013 Mar; 13: 819-820Crossref PubMed Scopus (85) Google Scholar, 4Cullinan N. Gorman K.M. Riordan M. Waldron M. Goodship T.H. Awan A. Case report: Benefits and challenges of long-term eculizumab in atypical hemolytic uremic syndrome.Pediatrics. 2015; 135: e1506-e1509https://doi.org/10.1542/peds.2014-3503Crossref PubMed Scopus (30) Google Scholar, 5Parikh SR, Lucidarme J, Bingham C, Warwicker P, Goodship T, Ramsay ME et al. First report of meningococcal B vaccine failure in a young adult on long-term eculizumab. 20th International Pathogenic Neisseria Conference, 4th–9th September 2016. Manchester, United Kingdom. http://www.ipnc2016.org/IPNC2016AbstractBook.pdf. Page 54.Google Scholar, 6Hernando Real S. Vega Castaño S. Pajares García R. Meningococcemia in vaccinated patient under treatment with eculizumab.Enferm Infecc Microbiol Clin. 2017 Mar; 35: 200-201https://doi.org/10.1016/j.eimCrossref PubMed Google Scholar] (including two events in the same patient [[6]Hernando Real S. Vega Castaño S. Pajares García R. Meningococcemia in vaccinated patient under treatment with eculizumab.Enferm Infecc Microbiol Clin. 2017 Mar; 35: 200-201https://doi.org/10.1016/j.eimCrossref PubMed Google Scholar]) (Table S1, online supplementary data). Of these, the presented case is the second described following the use of a conjugated meningococcal vaccine. Invasive disease is associated with a 10–40% fatality rate. Thus, prevention by population-based vaccination is considered a major strategy in the battle against this pathogen. Meningococcal vaccine preparations include a quadrivalent polysaccharide A,C,W-135,Y meningococcal vaccine, conjugated vaccines to A,C,W-135,Y, and a hybrid vaccine that confers immunity to C and Y meningococcal serogroups in addition to Haemophilus influenza type b. Recently, two recombinant preparations that protect against serogroup B were introduced. Although the quadrivalent polysaccharide and conjugated vaccines protect against the same serogroups, major differences in immunogenicity have been noted between these preparations. Conjugation is believed to activate T cells and induce immune memory, therefore conferring a longer protection and a reduced carriage rate. In four of the nine described cases, meningococcal sepsis was caused by a serogroup against which the patients were not vaccinated [1Hillmen P. Muus P. Röth A. Elebute M.O. Risitano A.M. Schrezenmeier H. et al.Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal hemoglobinuria.Br J Haematol. 2013; 162: 62-73Crossref PubMed Scopus (255) Google Scholar, 2Vicente D. Esnal O. Pérez-Trallero E. Fatal Neisseria meningitidis serogroup X sepsis in immunocompromised patients in Spain. Virulence of clinical isolates.J Infect. 2012 Feb; 64: 184-187Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 6Hernando Real S. Vega Castaño S. Pajares García R. Meningococcemia in vaccinated patient under treatment with eculizumab.Enferm Infecc Microbiol Clin. 2017 Mar; 35: 200-201https://doi.org/10.1016/j.eimCrossref PubMed Google Scholar]. In one of the five cases in which the vaccine did match the serogroup of invading bacteria, the non-conjugated polysaccharide vaccine was used, which is less immunogenic than the conjugated vaccine currently in use. Additionally, this patient was heavily immune suppressed and failed to mount a proper humoral response [[3]Struijk G.H. Bouts A.H. Rijkers G.T. Kuin E.A. ten Berge I.J. Bemelman F.J. Meningococcal sepsis complicating eculizumab treatment despite prior vaccination.Am J Transplant. 2013 Mar; 13: 819-820Crossref PubMed Scopus (85) Google Scholar]. Another patient was vaccinated with a conjugated vaccine, but featured suboptimal antibody titres [[4]Cullinan N. Gorman K.M. Riordan M. Waldron M. Goodship T.H. Awan A. Case report: Benefits and challenges of long-term eculizumab in atypical hemolytic uremic syndrome.Pediatrics. 2015; 135: e1506-e1509https://doi.org/10.1542/peds.2014-3503Crossref PubMed Scopus (30) Google Scholar], thus it is useful to measure serological response to meningococcal vaccine, and repeat vaccination every 3 years as suggested by Alashkar et al. Importantly, decreased susceptibility to penicillin reported ranging from 18% in 2010 in the USA to 55.7% in Tunisia, and the emergence of penicillin-resistant meningococcal strains, will render the common practice of administrating prophylactic penicillin treatment ineffective in an increasing number of cases. Of note, two of the nine described cases developed infection with a non-susceptible strains [4Cullinan N. Gorman K.M. Riordan M. Waldron M. Goodship T.H. Awan A. Case report: Benefits and challenges of long-term eculizumab in atypical hemolytic uremic syndrome.Pediatrics. 2015; 135: e1506-e1509https://doi.org/10.1542/peds.2014-3503Crossref PubMed Scopus (30) Google Scholar, 5Parikh SR, Lucidarme J, Bingham C, Warwicker P, Goodship T, Ramsay ME et al. First report of meningococcal B vaccine failure in a young adult on long-term eculizumab. 20th International Pathogenic Neisseria Conference, 4th–9th September 2016. Manchester, United Kingdom. http://www.ipnc2016.org/IPNC2016AbstractBook.pdf. Page 54.Google Scholar]. As conjugated meningococcal vaccines have been shown to reduce pharyngeal meningococcal carriage, we suggest a modified strategy that includes vaccination of close household contacts of eculizumab treatment candidates, coupled with routine surveillance aimed at identification of a Neisseria meningitidis carrier state among these contacts. Those found to be carriers of Neisseria meningitidis should be offered a targeted, susceptibility-based antibiotic course, in addition to the routine but increasingly ineffective penicillin prophylactic treatment. An alternative approach is providing patients with oral 750 mg ciprofloxacin, coupled with comprehensive explanation to use it when symptoms compatible with meningococcal infection occur, while seeking immediate medical evaluation. The following is the supplementary data related to this article: TABLE S1. Cases of IMD following prior vaccination, as described in the medical literature (including two events in the same patient) Download .docx (.02 MB) Help with docx files

Background: Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome. Methods: Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 mu m, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight. Findings: 2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients. Interpretation: Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

Background: Red blood cells (RBCs) undergo a natural aging process occurring in the blood circulation throughout the RBC lifespan or during routine cold storage in the blood bank. The aging of RBCs is associated with the elevation of mechanical fragility (MF) or osmotic fragility (OF) of RBCs, which can lead to cell lysis. The present study was undertaken to identify RBC properties that characterize their susceptibility to destruction under osmotic/mechanical stress. Methods: RBCs were isolated from freshly donated blood or units of packed RBCs (PRBCs) and suspended in albumin-supplemented phosphate-buffered saline (PBS). In addition, PRBCs were separated by filtration through a microsphere column into two fractions: enriched with rigid (R-fraction) and deformable (D-fraction) cells. The RBCs were subjected to determination of deformability, MF and OF, moreover, the level of cell surface phosphatidylserine (PS) and the stomatin level in isolated RBC membranes were measured. Results: In the RBC population, the cells that were susceptible to mechanical and osmotic stress were characterized by low deformability and increased level of surface PS. The OF/MF was higher in the R-fraction than in the D-fraction. Stomatin was depleted in destroyed cells and in the R-fraction. Conclusion: RBC deformability, the levels of surface PS, and membrane stomatin can be used as markers of RBC fragility.

Objective: The objective of the study was to gauge the effect of storage lesions on the dielectric response of red blood cells (RBC), in particular those processes linked to deformations of the cellular membrane known as the β-dispersion. Approach: The dielectric response of RBC suspensions, exposed to blood-bank cold storage, was studied using time-domain dielectric spectroscopy (TDDS) in the frequency range of 500 kHz up to 1 GHz. The measured dielectric processes are characterized by their dielectric strength (Δε) and relaxation time (τ). Changes in the dielectric properties of the RBC suspensions due to storage-related lesions were evaluated. For a quantitative characterization of RBC lesions, we measured the deformability of fresh and stored RBC as expressed by their elongation ratio (ER), which was achieved under a shear stress of 3.0 Pa. Main Result: The results show that the storage of RBC induced a statistically significant decrease of dielectric relaxation times. In addition, a sound correlation between the mean values of ER and the relaxation times was observed (Spearman’s correlation coefficient ρ  =  0.847). We draw the conclusion that those alterations in the relaxation time are induced by changes in the shape of the RBC that happen during cold-storage. Significance: The evolution of the β-dispersion of RBC opens new possibilities in the blood bank inventory management.

American Journal of HematologyVolume 92, Issue 9 p. E559-E560 E-ONLY ARTICLESFree Access Deformability of transfused red blood cells is a potent effector of transfusion-induced hemoglobin increment: A study with β-thalassemia major patients Gregory Barshtein, Gregory Barshtein Department of Biochemistry, Hebrew University Faculty of Medicine, Jerusalem, IsraelSearch for more papers by this authorNeta Goldschmidt, Neta Goldschmidt Department of Hematology, Hadassah University Hospital, Jerusalem, IsraelSearch for more papers by this authorAxel R Pries, Axel R Pries Charité-Universitätsmedizin Berlin, Berlin, GermanySearch for more papers by this authorOrly Zelig, Orly Zelig Blood Bank, Hadassah-Hebrew University Hospital, Jerusalem, IsraelSearch for more papers by this authorDan Arbell, Dan Arbell Department of Pediatric Surgery, Hadassah- Hebrew University Hospital, Jerusalem, IsraelSearch for more papers by this authorSaul Yedgar, Corresponding Author Saul Yedgar yedgar@md2.huji.ac.il orcid.org/0000-0001-7676-9017 Department of Biochemistry, Hebrew University Faculty of Medicine, Jerusalem, IsraelCorrespondence Saul Yedgar, Department of Biochemistry, The Hebrew University Faculty of Medicine, Jerusalem, Israel 91120. Email: yedgar@md2.huji.ac.ilSearch for more papers by this author Gregory Barshtein, Gregory Barshtein Department of Biochemistry, Hebrew University Faculty of Medicine, Jerusalem, IsraelSearch for more papers by this authorNeta Goldschmidt, Neta Goldschmidt Department of Hematology, Hadassah University Hospital, Jerusalem, IsraelSearch for more papers by this authorAxel R Pries, Axel R Pries Charité-Universitätsmedizin Berlin, Berlin, GermanySearch for more papers by this authorOrly Zelig, Orly Zelig Blood Bank, Hadassah-Hebrew University Hospital, Jerusalem, IsraelSearch for more papers by this authorDan Arbell, Dan Arbell Department of Pediatric Surgery, Hadassah- Hebrew University Hospital, Jerusalem, IsraelSearch for more papers by this authorSaul Yedgar, Corresponding Author Saul Yedgar yedgar@md2.huji.ac.il orcid.org/0000-0001-7676-9017 Department of Biochemistry, Hebrew University Faculty of Medicine, Jerusalem, IsraelCorrespondence Saul Yedgar, Department of Biochemistry, The Hebrew University Faculty of Medicine, Jerusalem, Israel 91120. Email: yedgar@md2.huji.ac.ilSearch for more papers by this author First published: 14 June 2017 https://doi.org/10.1002/ajh.24821Citations: 21AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In the current routine, blood donations are subjected to testing of blood type and pathogenic gents on day of donation, and the supply of packed red blood cells (PRBC) units for transfusion is conducted primarily according to storage duration, namely by the first-in-first-out (FIFO) criterion. However, the actual functionality of the transfused PRBC, namely their capacity to effect the expected transfusion outcome is ignored. This is especially pertinent to patients with chronic anemia, like β-thalassemia major (TM), who are treated with life-long frequent transfusions (of one or two units) every 2–4 weeks. The transfused RBC are aimed at raising the hemoglobin (Hb) level in the recipients' blood. However, RBC have unique mechanical properties, deformability in particular, which play a major role in blood circulation and in the RBC survival. In a recent study1 with TM patients, we have shown, for the first time in humans, that the transfusion-induced change in the recipients' skin blood flow strongly correlated with the deformability of the transfused PRBC. In the present study, we examined the effect of transfused RBC deformability on the immediate transfusion outcome, as expressed by the increase in the recipients' Hb (ΔHb), as well as the time interval between consecutive transfusions (TIBT). Employing TM patients for this study has specific advantages; TM patients are treated with life-long frequent transfusions (every 2–4 weeks). Therefore, testing the effect of repeated, consecutive transfusions in the same patient, having about the same baseline throughout the study, provides solid grounds for attributing the observed effect to the properties of the transfused PRBC units. In addition, TM patients at the Hadassah Hospital Thalassemia Clinic are routinely given PRBC units stored for up to about 10 days, when the potential storage-lesion is insignificant. Twenty-four TM patients were employed; their characteristics are summarized in Supporting Information Table S1. The transfusion-induced changes in the recipients' hemoglobin (ΔHb) and hematocrit (ΔHct) were determined 10 minutes after the transfusion completion. The results clearly showed that: ΔHb exhibited a highly significant positive correlation with RBC deformability: The transfusion outcome was analyzed vs. various parameters derived from deformability distribution of the PRBC. It was found that ΔHb, shown in Figure 1, as well as ΔHct (Supporting Information Table S2), were best correlated with the percent of low deformable cells (% LDFC), with highly significant inverse dependence on this parameter. Figure 1Open in figure viewerPowerPoint Transfusion-induced change in recipient's hemoglobin, ΔHb, per transfused PRBC unit, versus the percentage of low deformable cells (% LDFC) in the PRBC population. n = 51; r = −0.477; P = .0006 In addition to the immediate increase in the recipients' hemoglobin, an important criterion of the transfusion efficacy in TM patients is the time interval between consecutive transfusions (TIBT). As noted above, TM patients are treated with frequent (every 2–4 weeks) transfusions, and the longer is the TIBT, the better is the transfusion outcome. In the present study we have found that PRBC with low level of rigid RBC yields a longer interval between two consecutive transfusions (> 21 days), suggesting that RBC with good deformability would endure longer in the circulation and enable less frequent consecutive transfusions (Supporting Information Figure S2). While the transfusion of one PRBC unit is normally expected to induce ΔHb of 1 g/dL in the recipient's blood, in the TM patients, ΔHb, determined 10 minutes after transfusion, was 0.84 ± 0.40 g/dl following a one-unit transfusion, and 0.71 ± 0.29 g/dl (average) following a two-unit transfusion (Supporting Information Table S3). It thus seems that even in splenectomized TM patients, as employed in this study, the clearance of transfused PRBC is accelerated. Notably, dependence of ΔHb and ΔHct, determined upon completion of the transfusion, on the % LDFC, suggests that the relatively rigid cells in the transfused PRBC are cleared rapidly, practically during the transfusion procedure. This is in accord with previous reports that the clearance of transfused PRBC starts immediately with the administration of the PRBC into the blood stream.2 In splenectomized TM patients, as employed in the present study, the clearance of transfused RBC is expected to be slower. However, the opposite was observed here, as the transfusion-induced ΔHb was considerably lower than normally expected. Rigid and fragile RBC are prone to facilitated removal from the blood circulation within the first hours after transfusion, mainly due to the shear forces in the blood stream, in addition to splenic removal. In line with that, Nagababu et al.3 observed the elevation of plasma-free Hb immediately after PRBC transfusion. In addition, RBCs phagocytosis, in the spleen or liver, has been proposed to be the result of a balance between clearance enhancing ("eat me") signals, including phosphatidylserine (PS) on membrane surface and membrane band-3 clustering, and clearance-attenuating ("don't eat me") signals, such as CD44. In accord with that, we have shown that the rigid RBC are especially susceptible to mechanical stress, such as that applied by flow-induced shear stress, and are enriched with cell surface PS.4 These non-splenic routes may account for the accelerated clearance of transfused RBC in splenectomized TM patients, observed in the present study. As noted above, the supply of PRBC units for transfusion is conducted primarily according to the FIFO criterion, while the actual functionality of the transfused PRBC is ignored. In a preceding study with TM patients,1 we have shown that the transfusion-induced change in the recipients' skin blood flow was strongly correlated to the deformability of the transfused PRBC. In the present study we show, for the first time in humans, that the deformability of the transfused PRBC, in particular the % LDFC, is a major determinant of the transfusion-induced increase in the recipient's hemoglobin, the definitive goal of blood transfusion. Taken together, these studies strongly support the need for considering the hemodynamic functionality of transfused blood units in blood banking, which can be a powerful tool for reducing transfusion-related risks, especially for patients with chronic anemia, who are treated with frequent, long-life blood transfusions. The additional testing of the PRBC hemodynamic functionality will introduce a new concept into blood banking which will make a considerable contribution to improving transfusion therapy. CONFLICT OF INTEREST The authors have no conflict of interest to disclose. Supporting Information Additional Supporting Information may be found in the online version of this article. Filename Description ajh24821-sup-0001-suppinfo.docx70.4 KB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Barshtein G, Pries AR, Goldschmidt N, et al. Deformability of transfused red blood cells is a potent determinant of transfusion-induced change in recipient's blood flow. Microcirculation. 2016; 23: 479– 486. 2Bosman GJ. Survival of red blood cells after transfusion: processes and consequences. Front Physiol. 2013; 4: 376. 3Nagababu E, Scott AV, Johnson DJ, et al. The impact of surgery and stored red blood cell transfusions on nitric oxide homeostasis. Anesth Analg. 2016; 132: 274– 282. 4Orbach A, Zelig O, Yedgar S, et al. Biophysical and biochemical markers of red blood cells fragility. Transfus Med Hemother. 2017; 44: 183– 187. Citing Literature Volume92, Issue9September 2017Pages E559-E560 FiguresReferencesRelatedInformation

Background: Blood transfusions save lives and improve health; however, unnecessary transfusion practice exposes patients to immediate and long-term negative consequences. One of the 2013 Choosing Wisely®recommendations focused on avoiding liberal red blood cell (RBC) transfusion. The specific recommendation was that in situations where transfusion of RBCs is necessary, transfuse the minimum number of units required to relieve symptoms of anemia or to return the patient to a safe hemoglobin (Hb) range (7-8 gr/dl in stable, non-cardiac inpatients). As part of an initiative to improve the quality of RBC transfusion practice we performed this study to examine RBC transfusion practices by patient- and admission-related characteristics.

OBJECTIVE:There is a growing concern regarding the risks in the transfusion of PRBC, as numerous studies have reported negative transfusion outcomes, including reduced blood perfusion. In search of this phenomenon's mechanism, the effect of PRBC deformability, a major determinant of blood flow, on transfusion outcome was explored.METHODS:The effect of PRBC deformability was examined by the transfusion-induced change in recipients' ∆SBF, in β-TM patients, who are routinely treated with lifelong frequent transfusions. SBF was determined using a laser Doppler imager.RESULTS:∆SBF was examined vs PRBC deformability, the transfusion-induced increase in ∆Hct and the recipients' SBF before transfusion (SBFB ). ∆SBF elevated with increasing PRBC deformability, with a highly significant dependence, while its elevation with ∆Hct was much less significant. ∆SBF was inversely proportional to the SBFB .CONCLUSIONS:This study provides, for the first time in humans, direct evidence that the deformability of transfused PRBC is a potent effector of transfusion outcome. Currently, PRBC are supplied primarily by the first-in-first-out criteria, while their functionality is ignored. The testing of PRBC hemodynamic quality would introduce a new paradigm into blood banking, which would contribute substantially to improving transfusion therapy.