Clearance Of Red Cells Research Articles

Immunomodulatory roles of red blood cells.

To discuss recent advances supporting the role of red blood cells (RBCs) in the host immune response. Over the last century, research has demonstrated that red blood cells exhibit functions beyond oxygen transport, including immune function. Recent work indicates that the nucleic acid sensing receptor, toll-like receptor 9 (TLR9), is expressed on the RBC surface and implicated in innate immune activation and red cell clearance during inflammatory states. In addition to this DNA-sensing role of RBCs, there is growing evidence that RBCs may influence immune function by inducing vascular dysfunction. RBC proteomics and metabolomics have provided additional insight into RBC immune function, with several studies indicating changes to RBC membrane structure and metabolism in response to severe acute respiratory syndrome coronavirus 2 infection. These structural RBC changes may even provide insight into the pathophysiology of the 'long-coronavirus disease 2019' phenomenon. Finally, evidence suggests that RBCs may influence host immune responses via complement regulation. Taken together, these recent findings indicate RBCs possess immune function. Further studies will be required to elucidate better how RBC immune function contributes to the heterogeneous host response during inflammatory states. The appreciation for nongas exchanging, red blood cell immune functions is rapidly growing. A better understanding of these RBC functions may provide insight into the heterogeneity observed in the host immune response to infection and inflammation.

How Do Red Blood Cells Die?

Normal human red blood cells have an average life span of about 120 days in the circulation after which they are engulfed by macrophages. This is an extremely efficient process as macrophages phagocytose about 5 million erythrocytes every second without any significant release of hemoglobin in the circulation. Despite large number of investigations, the precise molecular mechanism by which macrophages recognize senescent red blood cells for clearance remains elusive. Red cells undergo several physicochemical changes as they age in the circulation. Several of these changes have been proposed as a recognition tag for macrophages. Most prevalent hypotheses for red cell clearance mechanism(s) are expression of neoantigens on red cell surface, exposure phosphatidylserine and decreased deformability. While there is some correlation between these changes with aging their causal role for red cell clearance has not been established. Despite plethora of investigations, we still have incomplete understanding of the molecular details of red cell clearance. In this review, we have reviewed the recent data on clearance of senescent red cells. We anticipate recent progresses in in vivo red cell labeling and the explosion of modern proteomic techniques will, in near future, facilitate our understanding of red cell senescence and their destruction.

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77–81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57–83% but 15–58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.

Abstract 3256: RTX-IL-12, an allogeneic red cell therapeutic expressing IL-12, exhibits potent in vitro and in vivo activity and favorable safety profile

Abstract Recombinant IL-12 is a potent cytokine that has held significant promise as an immunotherapeutic. In preclinical studies, recombinant IL-12 has impressive anti-tumor activity; however toxicity and a narrow therapeutic window halted development in humans. To address these limitations, Rubius Therapeutics has developed genetically engineered red cell therapeutics (RCTs) with cell surface expression of IL-12 alone (RTX-IL-12) or in combination with co-stimulatory or other cytokine molecules. These cells present ligands in their native form that potently activate and expand both T and NK cells through potent cell-cell interactions. On-target, off-tissue toxicity of RCTs may be limited due to their biodistribution, which is restricted to the vascular system. Based on its reported role in promoting a TH1 response and proliferation of cytotoxic NK and CD8 T cells, RTX-IL-12 activity was evaluated in vitro and demonstrated proliferation of human CD8 T cells (2-3 fold), Th1 differentiation of naïve CD4 T cells, IFNγproduction (6-11 fold increase over control), as well as NK cell activation and cytotoxicity against K562 targets (2-5 fold increase over control). To evaluate in vivoimmune response, anti-tumor activity and safety, a mouse surrogate red cell therapeutic, mRBC-IL-12, was developed by chemically conjugating recombinant Fc-IL-12 to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Administration of mRBC-IL-12 (1X109cells) to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 52% decrease in the number of lung metastases and was associated with increased proliferating and cytotoxic CD8 and NK cells in the lungs (p=0.0002). Administration of mRBC-IL-12 (1X109cells) to mice bearing B16F10 and MC38 subcutaneous models exhibited 82% and 80% tumor growth inhibition, respectively. When combined with anti-PD1 treatment, mRBC-IL-12 (1X109cells) efficacy was improved in these two models and showed 86% and 85% tumor growth inhibition, respectively. Efficacy was accompanied by an increase in survival (p=0.0004 B16F10, p=0.0003 MC38 ) and the infiltration of M1 macrophages (p=0.007) into the tumor. Mice treated with the highest feasible dose of mRBC-IL-12 displayed no significant body weight loss and 3-10-fold lower serum IFNg levels compared to soluble rec. Fc-IL-12. By combining IL-12 with IL-15TP or 4-1BBL on the same red cell, tumor growth was strongly inhibited and in some cases improved over mRBC-IL-12 alone (TGI 46% and 63% in B16F10 SC, TGI 83%, 77% in MC38 and 78%, 70% in B16F10 IV). In summary, the data demonstrate that sequestering IL-12 in the vasculature through expression on red cells drives significant reductions in tumor growth, while improving the tolerability profile of the cytokine,supporting further testing in cancer patients. Citation Format: Anne-Sophie Dugast, Enping Hong, Maegan Hoover, Arjun Bollampalli, Douglas C. McLaughlin, Omkar Bhate, Timothy J. Lyford, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-IL-12, an allogeneic red cell therapeutic expressing IL-12, exhibits potent in vitro and in vivo activity and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3256.

Abstract 3272: RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile

Abstract Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical activity perhaps due to 1) toxicity; 2) a need for coordinated activation of co-stimulatory and cytokine pathways; or 3) the failure of agonist antibodies to recapitulate signaling by endogenous ligands. To address these limitations, Rubius Therapeutics developed genetically engineered red cells with cell surface expression of both co-stimulatory and cytokine ligands, which present ligands in their native form through cell-cell contact to potently activate and expand both T and NK cells. On-target, off-tissue toxicity may be limited due their biodistribution, which is restricted to the vascular system. IL-15 is known to promote NK survival and CD8 T cell memory and 4-1BB agonists are known to promote T cell prolioferation and survival. Rubius Therapeutics has developed RTX-212, an allogeneic red cell therapeutic genetically engineered to co-express 4-1BBL and an IL-15/IL-15Rαfusion (IL-15TP). In vitro assays demonstrated that the combination of both ligands on RTX-212 expanded both memory CD8 T cells (2.5-fold) and NK cells (10-15-fold), in the absence of TCR stimulation. RTX-212 further induced dramatic proliferation of CD8 T cells and CD4 T cells in the presence of TCR stimulation with increased IFNγsecretion. In addition to the synergistic effects of 4-1BBL and IL-15TP, each molecule provided complementary functions that expanded the activity of RTX-212 beyond RTX-4-1BBL or RTX-IL-15TP alone. IL-15TP uniquely activated NK cytotoxicity and ADCC, while 4-1BBL uniquely stimulated CD4 and CD8 T cell proliferation and production of IFNγ. To evaluate in vivo immune responses, anti-tumor activity and safety, a mouse surrogate therapeutic, mRBC-212, was developed where recombinant Fc-IL-15-sushi and m4-1BBL were chemically conjugated to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Intravanous administration of mRBC-212 to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 66% decrease in the number of lung metastases compared to control mice (p=0.0001) and was associated with a significant increase in NK cell infiltration into the lungs (p=0.02). In a CT26 tumor model, mRBC-212 treated mice exhibited 55% tumor growth inhibition, which was accompanied by a 1.7-fold increase in the tumor infiltration of proliferating and cytotoxic CD8 T cells. Mice treated with the highest feasible dose of mRBC-212 showed no change in serum transaminases, infiltration of CD8 T cells and macrophages to the liver or liver inflammation score compared to agonistic 4-1BB antibodies treated mice. Taken together, these data indicate that RTX-212 has the potential to be an effective therapy with an improved safety profile compared to 4-1BB agonist antibodies and IL-15 agonists, supporting its clinical development. Citation Format: Anne-Sophie Dugast, Shannon McArdel, Maegan Hoover, Enping Hong, Shannon Curtis Leonard, Arjun Bollampalli, Douglas C. McLaughlin, Jennifer Mellen, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3272.

A role for red cell clearance in antibody‐mediated inhibition of erythrocyte alloimmunization?

Antibodies targeting erythrocytes can induce antibody‐mediated inhibition of erythrocyte alloimmunization, and anti‐D has been extremely successful in preventing haemolytic disease of the foetus and newborn (HDFN). It is desirable to replace the current donor‐derived anti‐D with a monoclonal antibody; however, the exact mechanism of IgG‐mediated suppression of red blood cell immune responses remains unclear. It has been proposed that the ability of anti‐D to prevent HDFN is due to IgG interactions with Fc receptors on phagocytic cells leading to rapid clearance of RhD+ red cells. Several monoclonal anti‐D alternatives have been developed with an emphasis on their ability to rapidly clear red blood cells from the circulation. None of the monoclonal antibodies have been as effective as donor‐derived anti‐D with some antibodies inadvertently leading to immune enhancement instead of inhibition. A better understanding of the mechanisms of IgG‐mediated inhibition of red cell alloimmunization is necessary. In this brief review, we highlight selected evidence for and against the requirement for rapid red cell clearance in the ability of IgG to prevent red cell alloimmunization. We also discuss potential alternative mechanisms which could be important for informing the future development of monoclonal antibody alternatives.

What can we learn from variation of red cell size distribution?

What can we learn from variation of red cell size distribution?

Physics vs Biology of Phagocytosis: Cell Rigidity and Shape Override CD47 ‘Self’ Signaling in Phagocytosis by Hyperactivating Myosin-II

A macrophage engulfs another cell or foreign particle in an adhesive process that often activates Myosin-II, unless the macrophage also engages ‘Marker of Self’ CD47 that inhibits Myosin. Adhesion processes of many cell types also activate Myosin and increasingly so on rigid substrates. Here we demonstrate that the rigidity of a phagocytosed RBC hyperactivates Myosin to overwhelm ‘Self’ signaling to macrophages. RBC stiffness is one among many factors, including shape, that change in senescence, some anemias, and diseases such as malaria. Controlled stiffening of normal human RBCs in different shapes did not compromise interactions of CD47 on RBCs with the macrophage ‘Self’-recognition receptor, SIRPa. Uptake of antibody-opsonized RBC was always fastest with rigid RBC-Discocytes, which also showed maximal active Myosin at the phagocytic synapse that biophysically out-competed ‘Self’ signaling by CD47. Rigid but more rounded RBC-Stomatocytes signaled ‘Self’ moreso than rigid RBC-Discocytes, highlighting the effects of shape. Physical properties of phagocytic targets can thus modulate ‘Self’ signaling as seems relevant to splenic clearance of rigid red cells after storage or clearance of rigid pathological cells such as sickle and thalassemic red cells.

Antibody-mediated immune suppression of erythrocyte alloimmunization can occur independently from red cell clearance or epitope masking in a murine model.

Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

Regulation of red cell life-span, erythropoiesis, senescence, and clearance.

The number of red blood cells (RBCs) and their properties are optimized by nature for most efficient oxygen delivery from the lungs to hypoxic periphery. Changes in metabolic requirements or environmental oxygen availability quickly translate to modulation of the RBC number, blood rheology, oxygen affinity of hemoglobin, and even of vascular tone. Inability to match the changes in oxygen demand may be fatal and requires therapeutic intervention. The recent advances in the ongoing intensive investigations of the mechanisms in control of regulation of erythropoiesis, RBC maturation and aging, as well as the processes involved in recognition of senescent RBCs and their clearance make up the present volume. It all starts from within the mesoderm, the fetal liver and from the adult bone marrow where primitive or definitive erythropoiesis takes place (Palis, 2014). Facilitated RBC production may be induced promptly “on demand” in extended oxygen requirements upon ascent to the high altitude and quickly reversed when extra RBC mass is without benefit any more (Risso et al., 2014). Exercise and professional sport increase RBC turnover and maximize oxygen delivery to the tissues (Mairbaurl, 2013). Maturation and aging of RBCs is accompanied by multiple processes occurring at various rates driving the circulating RBCs from adolescence to senescence within approximately 120 days (Lew and Tiffert, 2013; Lutz and Bogdanova, 2013). The resulting “markers of senescence” are recognized by the macrophages and clearance of RBCs is promptly initiated (de Back et al., 2014). Premature clearance is a hallmark of various disorders associated with anemia. In each case one or multiple markers of senescence appear prematurely. Those include excessive oxidative stress (Mohanty et al., 2014), excessive cation leak with the following dehydration (Wang et al., 2014), decrease in RBC size and loss of RBC membrane through vesiculation (Alaarg et al., 2013), metabolic abnormalities (Vives-Corrons et al., 2013), or following auto-immune diseases (Lutz and Bogdanova, 2013). Blood storage damages RBCs facilitating aging. As a result clearance of transfused cells is dramatically facilitated (Bosman, 2013; Flatt et al., 2014). The present compilation does not only give an overview of the variety of opinions reflecting the current understanding of the mechanisms of erythropoiesis, aging, and clearance of RBCs. We hope that it also provides the base for future lively discussions of the up-standing problems in this rapidly developing research area.

Hemolysis After High Dose IVIG In Blood Group A Women With Alloimmune Thrombocytopenia

IntroductionHemolysis after IVIG infusion has been observed in clinical trials and case reports. Certain factors seem to place a patient receiving IVIG at higher risk for hemolytic anemia: 1) having blood type A, B, or AB and 2) receiving high doses of IVIG (≥ 2g/kg).IVIG contains isohemagglutinins that coat red blood cells and may cause red cell clearance by the RES (Table 1). Anti-A, Anti-B, and Anti-D titers have been detected in IVIG products at varying concentrations by different IVIG manufacturers. It has been suggested that Anti-A titers > 1:16 are more likely to cause clinically significant hemolysis and thus anemia in recipients.Table 1Anti-A Titer Levels by IVIG PreparationIVIG BrandAnti-A TiterGammagard liquid1:64Privigen 10%1:64Gamunex 10%1:32Octagam 10%1:8 - 1:32Gamimune-N1:8Octagam 5%1:2 - 1:8Carimune 9%1:4Table 2IVIG-Associated Hemolysis by Blood Type (BT)Blood TypePercent Experiencing HemolysisBlood Type Prevalence in USA61.9%40%B17.5%11%AB12.7%4%O4.8%45%15 summarized studies reported 63 patients who experienced hemolysis after IVIG infusion and showed the higher risk of non-group O recipients to hemolyze with IVIG:In the current study, we sought to explore the degree of anemia as related to ABO blood types in mothers treated with IVIG for fetal alloimmune thrombocytopenia (AIT). MethodsA retrospective chart review was conducted on 84 women who had received IVIG for AIT treatment during their pregnancy to increase the fetal platelet count. Women were randomized to receive IVIG 2g/kg/wk (arm A, n=43) or IVIG 1g/kg/wk + prednisone (arm B, n=41) starting at 20-30 weeks of gestation until delivery. One CBC per month was collected from each mother from the start of treatment until delivery. Hemoglobins and mean corpuscular volumes (MCVs) were tracked and development of anemia was compared among women with blood types (BT) A, B, AB and O in each treatment arm. Patients who non-randomly received IVIG 2g/kg/wk + prednisone together as rescuetreatment were excluded from this study. Results36 of the 84 women had hemoglobins less than 10 on at least one CBC during IVIG treatment. All 36 had MCV values in the normocytic range (80-100). The degree of anemia was significantly greater in Arm A (IVIG 2g/kg) than Arm B (IVIG 1g/kg + pred) (Fisher's exact test, p=0.016; table 3).Table 3Degree of Hemolysis among treatment armsArm A (IVIG 2g/kg/wk)Arm B (IVIG 1g/kg/wk + Prednisone 0.5 mg/kg/d)Hg<102412Hg≥101929Among women in arm A (IVIG 2g/kg), BT non-O had a significantly higher incidence of marked anemia (Hg<10) than those with BT O (Fisher's exact test, p=.001; table 4), with BT A having the highest number of women with Hg<10 (A=16, B=1, AB=1, O=4, unknown=2).There was no significant difference among women by blood type O (n=4) versus non-O (A=4,B=2,AB=0,unknown=2) in the incidence of anemia in arm B (IVIG 1g/kg + pred); both BT groups showed a low incidence of hemolysis (table 5).Table 4IVIG 2g/kg/wk (Arm A)Blood type OBlood type Non-OHg<10420Hg≥10136Table 5IVIG 1g/kg/wk + Pred (Arm B)Blood type OBlood type Non-OHg<1048Hg≥101415 ConclusionsPatients who were on 2g/kg/wk IVIG became substantially more anemic than those on the 1g/kg/wk IVIG + prednisone dose, which supports reported findings that higher dose (HD) IVIG is associated with a greater risk of anemia. Note: receiving IVIG 2g/kg/wk for multiple weeks is very high dose IVIG. In particular, those patients receiving HD IVIG who were BT A had a greater risk of developing a greater extent of anemia than those of blood type O. This finding is consistent with the hypothesis that blood-group specific antibodies in IVIG result in immune hemolysis primarily in BT A recipients because of the higher anti-A titers. Furthermore, while 1 g/kg/wk of IVIG is not “low dose”, the concomitant use of prednisone 0.5mg/kg/d may protect against development of a greater degree of anemia in these patients.IVIG is the standard of care treatment for AIT; however 1 g/kg/wk is known not sufficiently effective in severely thrombocytopenic fetuses (Berkowitz, OB-GYN 2006). Therefore we believe appropriate treatment of women whose previous babies did not have an ICH needs to be > 1g/kg/wk. Our study found that hemolytic anemia in non-O blood types may be an important side effect of 2g/kg/wk IVIG treatment, indicating that patient blood type should be considered when deciding which IVIG regimen to use. Disclosures:Bussel:Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; IgG of America: Research Funding; Genzyme: Research Funding; Cangene: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity's Board of Directors or advisory committees.

Red cell indices in classification and treatment of anemias

Measurements of red cell volume, hemoglobin (Hb) concentration and Hb content continue to play a crucial role in the differential diagnosis of anemias 80 years after the publication of Wintrobe's seminal work. Modern hematology analyzers provide additional data on the heterogeneity of these parameters (distribution width) and quantify similar parameters of reticulocytes as well. Red cell and reticulocyte cellular indices are widely used in the diagnosis and monitoring of hematological diseases. Quantification of hypochromic cells is valuable in the differential diagnosis of thalassemia trait and iron deficiency, and in monitoring therapeutic response to erythropoietic stimulating agents, while hyperchromic cells are an essential diagnostic component for hereditary spherocytosis and may correlate with hemolytic parameters in sickle cell disease. Values for these parameters however depend on the technology used. Red cell clearance is associated with a reduction in both Hb content and cell volume: normal cells are likely to be removed by the time they reach a volume of 72 fl. Reticulocyte parameters such as Hb content (CHr or ret-He) or maturity index (RMI) have shown value in a variety of hematological conditions. New findings from genetic association studies have identified several potential novel genes affecting red cell indices, which are not mediated by changes in iron availability. Red cell indices continue to provide an essential support to the diagnosis and monitoring of hematological diseases.

Sphingomyelinase-Induced Remodeling of the Erythrocyte Membrane

Abstract 1032Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, a major lipid component of cell membranes, generating phosphocholine and ceramide. Inflammation arising from various diseases including sepsis is known to enhance SMase secretion in the circulation. Also, chronic inflammation typically induces anemia through reduced production and enhanced clearance of red cells, which is often associated with poor disease outcome. Incubation of red cells with plasma of septic patients with enhanced SMase activity has been described to result in ceramide formation and exposure of phosphatidylserine (PS). Therefore, we studied the effect of SMase on various pathophysiologically relevant parameters of red cell homeostasis in fresh and stored erythrocytes.Using time-lapse confocal microscopy we observed a typical sequence of events during SMase treatment of erythrocytes. The cells first lost their discoid shape, PS became externalized, and ultimately there was a sudden loss of cytoplasmic content. Additional confocal microscopy, flow cytometry and immunoblot analyses showed that SMase treatment resulted in ceramide-induced alterations in red cell membrane-cytoskeleton interaction and organization, including lipid raft formation. These changes probably underlie the increase in osmotic fragility, membrane vesiculation and invagination, as well as large hemoglobin aggregates that we also observed. The membrane lipid scrambling, altered morphology, enhanced fragility and presence of rigid membrane patches may all contribute to enhanced removal of sphingomyelinase-exposed red cells.Generation of very low ceramide levels in the membrane by SMase, as detected by Positive-Ion MALDI-TOF MS, already affected red cell shape, membrane rearrangement and osmotic responsiveness. Combined with the observation that 24 hour incubation of red cells with pathological SMase levels induced shape change and PS exposure, these data suggest that red cell homeostasis could well be disturbed by SMase during chronic inflammation. Of note, red cell storage greatly increased the sensitivity to SMase-induced changes. This may well have implications for red cell transfusion in critically ill patients. Disclosures:No relevant conflicts of interest to declare.

Failure of eculizumab to correct paroxysmal cold hemoglobinuria

Dear Editor, Paroxysmal cold hemoglobinuria (PCH) is a rare cause of intravascular hemolytic anemia in which an antibody against the P antigen develops and hemolysis ensues via a pathway involving thermally dependent complement binding. Previously an association of syphilis due to crossreactivity of antibody, PCH has since predominantly become a disease of childhood following varicella-zoster virus infection. The childhood form is typically acute, selflimiting, and steroid-responsive. Rare cases of PCH in adults have been described in association with chronic lymphocytic leukemia and lymphoma but never previously with myeloma [1]. We describe a case of steroid-refractory PCH in a patient with myeloma that was only partially corrected with eculizumab, a monoclonal antibody against C5 complement. A 51-year-old Nigerian man with Stage III ISS IgA kappa multiple myeloma in very good partial response following treatment with bortezomib, thalidomide, cyclophosphamide, and dexamethasone was admitted to our hospital with severe hemolytic anemia. At admission, lactate dehydrogenase (LDH) was elevated at 543 U/L (100–200 U/L); bilirubin, 164 μmol/L (<20 μmol/L); and hemoglobin was 60 g/L (125–175 g/L). Initial transfusion requirement were 2 to 4 U of red blood cells per day to achieve a target hemoglobin 60–80 g/L. PCH was diagnosed with the Donath–Landsteiner test following positive urinary hemosiderin on day 21 of admission. No complement C3 was detected on his red cells at diagnosis. Blood film was significant for rosette formation of erythrocytes around neutrophils and erythrophagocytosis by neutrophils (Fig. 1). Syphilis serology and cerebrospinal fluid treponemal PCR were negative. Being refractory to oral prednisolone and cyclophosphamide (1 g/m), he received standard induction with eculizumab 600 mg weekly for 4 weeks [2]. Urinary hemosiderin became negative by day 36; however, hemolysis continued albeit at reduced rate, and was finally controlled following a single dose of intravenous cyclophosphamide 4 g/m. The patient became transfusionindependent 31 days post high-dose cyclophosphamide and hemoglobin returned to normal reference range (136 g/L) at 10 weeks, with normalization of LDH at 9 weeks. This is the first reported case of eculizumab use for treatment of PCH. Failure of eculizumab to control hemolysis in this patient suggests that extravascular clearance of red cells in PCH may be induced by earlier components of complement and opsonization with anti-P antibodies. Moreover, the observation of neutrophil erythG. P. Gregory (*) : S. Opat :H. Quach : J. Shortt :H. Tran Department of Clinical Hematology, Monash Medical Centre, Clayton, VIC, Australia e-mail: g.gregory@alfred.org.au

Eculizumab prevents intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low-level extravascular hemolysis occurring through C3 opsonization

Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by intravascular hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system. Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of hemolysis. Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test (P<0.01). Among the eculizumab-treated patients, the mean hemoglobin value for the 21 with a positive direct antiglobulin test was 9.6+/-0.3 g/dL, whereas that in the ten patients with a negative test was 11.0+/-0.4 g/dL (P=0.02). These data demonstrate a previously masked mechanism of red cell clearance in paroxysmal nocturnal hemoglobinuria and suggests that blockade of complement at C5 allows C3 fragment accumulation on some paroxysmal nocturnal hemoglobinuria red cells, explaining the residual low-level hemolysis occurring in some eculizumab-treated patients.

Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti‐D immunoglobulin: more questions than answers

Prophylactic anti-D is a very safe and effective therapy for the suppression of D-immunization and prevention of haemolytic disease of the foetus and newborn. The primary mode of action of anti-D is rapid clearance of fetal D-positive red cells from the maternal circulation, mediated by interactions with immunoglobulin G Fc receptors on macrophages in the spleen. Many anti-D monoclonal antibodies (mAb) have been produced by a variety of methods. Twelve anti-D mAbs were tested in eight studies for their ability to mediate clearance of autologous red cells, and 13 antibodies studied in seven trials of the clearance of D-positive red cells injected into D-negative subjects. Antibodies produced by human B-cell lines, mouse-human heterohybridomas and Chinese hamster ovary cells varied in their activity with none being quite as effective as polyclonal anti-D. However, clearance mediated by recombinant anti-D produced by rat YB2/0 cells was extremely rapid, faster than polyclonal anti-D, but with haemolysis and some hepatic accumulation of red cells observed in one study. Two human anti-D mAbs prevented D-immunization. In contrast, anti-D mAbs from heterohybridomas increased the incidence and rapidity of anti-D responses. It is hypothesised that unnatural glycosylation of monoclonal anti-D produced by some cell lines may have caused these unexpected results. In some antibodies, unusual oligosaccharides on anti-D may have affected binding to Fc receptors resulting in reduced red cell clearance. For others, non-human glycoforms of anti-D might have bound to innate immune recognition molecules promoting pro-inflammatory reactions. These extensive data on the clinical activity of monoclonal anti-D produced by cell lines derived from four species will inform the future development of monoclonal anti-D for RhD prophylaxis.

Species- and cell type-specific interactions between CD47 and human SIRPα

AbstractCD47 on red blood cells (RBCs) reportedly signals “self” by binding SIRPα on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRPα1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRPα1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRPα-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRPα1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRPα1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRPα1 significantly. The results thus demonstrate that SIRPα-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity. (Blood. 2006;107:2548-2556)

Monoclonal anti-D antibodies to prevent alloimmunization: lessons from clinical trials

In the past 20 years, numerous monoclonal anti-D antibodies have been developed in order to replace the human plasma derived anti-D immunoglobulins, using different in vitro functional assays as screening methods. Some of these monoclonal antibodies have been evaluated in exploratory in vivo clinical trials, notably for their ability to mediate the clearance of D-positive red cells. A review of these reported trials is presented and the results are analyzed in the light of the newly published hypothesis conferring an important role to some Fc-FcγR interactions and to the glycosylation-dependent potency of the monoclonal anti-D antibodies.

The Role of Lactadherin in the Phagocytosis of Phosphatidylserine-Expressing Sickle Red Blood Cell by Macrophages.

Sickle cell anemia, the most common serious hemoglobinopathy, is associated with a markedly reduced life span of red blood cells due to their preferential clearance by macrophages. During polymerization of sickle hemoglobin, phosphatidylserine, an anionic phospholipid normally present exclusively on the inner leaflet of the membrane bilayer is exteriorized to outer leaflet. This exposure of phosphatidylserine is thought to be a tag for macrophage recognition. Lactadherin, also known as milk fat globule-EGF factor 8, is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of apoptotic cells. Here, we investigated the role of lactadherin in the phagocytosis of sickle red blood cells.The binding of fluorescein-lactadherin to normal and sickle red blood cells was studied by flow cytometry. We quantified the effect of lactadherin on phagocytosis of red blood cells by monocyte-derived macrophage.In normal individuals, less than 0.5% of red blood cells showed any binding to lactadherin when analyzed by flow cytometry. However, in sickle cell patients, circulating red blood cells showed 2 to 10- fold increase in lactadherin binding (P<0.0002). Lactadherin stimulated the phagocytosis of resting sickle red blood cells by macrophages but had no significant effect on the phagocytosis of normal red blood cells. Deoxygenation of sickle red blood cells further increased the lactadherin binding and phagocytosis. Antibodies to integrin αVβ3 also inhibited macrophage binding and phagocytosis.These results show lactadherin may play a major role in sickle red cell clearance by anchoring the phosphatidylserine-expressing sickle red blood cells to integrins on tissue macrophages.

The complexities of the Rh system.

In 1939, Levine & Stetson first described the case of a mother, after giving birth to a stillborn child, having a haemolytic transfusion reaction following transfusion her husband’s blood. Her serum agglutinated her husband’s red cells and those of 80% of ABO-compatible donors. In 1940, Landsteiner & Wiener injected Rhesus-monkey red blood cells into rabbits. The rabbit serum agglutinated Rhesus-monkey red cells, and also 85% of human red cells. They called this antibody anti-Rh, and in 1941 Levine & Stetson’s human antibody was shown to have the same pattern of reactivity as the rabbit anti-Rh antibody. However, by 1942, Fisk & Foord had demonstrated a difference between rabbit and human anti-Rh: red cells from all newborn babies, whether Rh positive or negative as defined by human anti-Rh were positive with the rabbit anti-Rh. In 1963, Levine et al . finally proved that human and rabbit anti-Rh did not react with the same antigen. However, owing to common usage of the term, ‘Rh’ was kept as the title for the human antibodies. The rabbit ‘anti-Rh’ was then called anti-LW in honour of Landsteiner & Wiener. Even today people often make the mistake of referring to the ‘Rhesus’ blood group system; this is incorrect, as the blood-group antigen has nothing to do with Rhesus monkeys, and should be referred to as the ‘Rh’ blood group system. In 1943, Wiener demonstrated the presence of six alleles in the Rh system with three different antisera, and since then the system has been shown to have many further variants and complexities. In 1953, it was discovered that some RhD-positive individuals can make anti-D, and this led to the hypothesis that the RhD antigen is a complex antigen comprised of a series of epitopes. A lack of part of the antigen means that an individual can make antibody to this part. In 1942, Levine confirmed that Rh incompatibility between mother and fetus was the major cause of haemolytic disease of the newborn (HDN). Various studies showed that, depending on the population, 3–25% lack the Rh antigen D, and that RhD-negative individuals readily make anti-D. RhD is thus of critical importance in blood transfusion, and all transfusions are routinely matched for RhD compatibility. In emergency situations RhD-negative blood is given, particularly if the recipient is a female of child-bearing age, to avoid immunization. RhD-negative women can be treated with prophylactic anti-D to prevent HDN. Although the mechanism of action for this process is not fully understood, it is apparent that rapid clearance of fetal red cells from the maternal circulation by passively administered anti-D prevents the mother making immune anti-D. A very successful programme of routine postdelivery administration of anti-D to RhD-negative mothers bearing RhD-positive babies has greatly reduced the incidence of HDN. However, some fetal cells can also enter the maternal bloodstream during pregnancy, and cause immunization. To prevent this cause of HDN, routine antenatal administration of anti-D is now being adopted in many countries.