Degradation Of Red Blood Cells Research Articles

Novel concepts in red blood cell clearance.

Red blood cell (RBC) clearance has been studied for decades in many different pathologies, which has revealed different routes of RBC degradation, depending on the situation. This review summarizes the latest mechanistic insights on RBC clearance in different contexts; during homeostatic removal, immune-mediated destruction, and systemic inflammation. Besides the recognition of a variety of potential 'eat me' signals on RBCs, recent evidence suggests that normal RBC degradation is driven by the increase of the adhesive properties of RBCs, mediating the retention in the spleen and leading to RBC hemolysis. Furthermore, immune-mediated degradation of RBCs seems to be fine-tuned by the balance between the density of the antigens expressed on RBCs and the presence of 'don't eat me' signals. Moreover, besides RBC clearance by macrophages, neutrophils seem to play a much more prominent role in immune-mediated RBC removal than anticipated. Lastly, RBC clearance during systemic inflammation appears to be driven by a combination of extreme macrophage activity in response to proinflammatory cytokines as well as direct damage of RBC by the inflammation or inflammatory agent. Recent studies on RBC clearance have expanded our knowledge on their destruction in different contexts.

A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12–24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.

Storage of blood clots for histological analysis: How long is too long in saline and paraformaldehyde?

To investigate the composition of blood clots following mechanical thrombectomy, it is essential to ensure optimum storage for highest quality histological and immunofluorescence analysis. We investigated for how long clots can be stored in paraformaldehyde (PFA), saline and heparinised saline before the tissue integrity is compromised. Whole blood and fibrin-rich clot analogues were made under dynamic flow conditions. Clots were stored in 4% PFA, saline or heparinised saline for timepoints ranging from 1 hour to two months. Five µm sections were stained with Martius Scarlet Blue to visualise red blood cells (RBCs), white blood cells (WBCs) and fibrin. Semi-quantitative analysis of the integrity of clot components used a scoring system (0: Poor; 1: Sub-par; 2: High). Quantitative analysis used Orbit Image Analysis software. Autofluorescence was assessed using a relative scale. Clots stored in PFA for up to two months were qualitatively similar to those stored for all shorter periods (median score: 2 per component). Clots stored in saline/heparinised saline for one week showed degradation of RBCs and WBCs, but fibrin remained intact (median score: 1, 1, 2 respectively). Degradation of the samples stored in saline/heparinised saline made accurate quantification using Image Analysis software difficult from 24h. Samples stored in PFA for up to two weeks showed an edging autofluorescence effect, which became more evident with prolonged storage. For optimum histology, ideally clots should not be stored in saline before fixation and should ideally be stored in formalin for less than one month to minimise the impact of autofluorescence on immunofluorescence.

Identification of Oxidative Stress in Red Blood Cells with Nanoscale Chemical Resolution by Infrared Nanospectroscopy.

During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Stiffness increase of red blood cells during storage

In transfusion medicine, the deformability of stored red blood cells (RBCs) changes during storage in blood banks. Compromised RBC deformability can reduce the transfusion efficiency or intensify transfusion complications, such as sepsis. This paper reports the microfluidic mechanical measurement of stored RBCs under the physiological deformation mode (that is, folding). Instead of using phenomenological metrics of deformation or elongation indices (DI or EI), the effective stiffness of RBCs, a flow velocity-independent parameter, is defined and used for the first time to evaluate the mechanical degradation of RBCs during storage. Fresh RBCs and RBCs stored up to 6 weeks (42 days) in the blood bank were measured, revealing that the effective stiffness of RBCs increases over the storage process. RBCs stored for 1 week started to show significantly higher stiffness than fresh RBCs, and stored RBC stiffness degraded faster during the last 3 weeks than during the first 3 weeks. Furthermore, the results indicate that the time points of the effective stiffness increase coincide well with the degradation patterns of S-nitrosothiols (SNO) and adenosine triphosphate (ATP) in RBC storage lesions.

L-Asparaginase Loaded Inside Red Cells Has An Acceptable Tolerability Profile On Bilirubin Value

L-asparaginase has been a major drug in the treatment of acute lymphoblastic leukemia (ALL) over the past decades, its efficacy has been demonstrated in a broad range of patient clinical profiles. However frequence and severity of toxicities, of which allergic reactions, have been a major drawback. L-asparaginase loaded inside homologous red cells, suspended in SAG-mannitol preservative solution, (GRASPA) is a new available formulation for administering L-asparaginase in combination with standard chemotherapy in different ALL populations, including frail patients older than 55years. Tolerability and efficacy of GRASPA in patients with ALL have been investigated in successive clinical trials (GRASPALL 2005-01 and GRASPALL/GRAALL-SA2-2008). The global toxicity of this new L-asparaginase formulation is decreased, in all age groups, with fewer hypersensitive reactions. A significant reduction of the coagulation disorders in patients <55 years old has also been reported. The pharmacokinetics is improved, with a half-life of about 30 days compared to 1 day for the free formulation. GRASPA has been proposed as a new approach to maintain the complete activity of L-asparaginase while reducing its antibody mediated toxicity. The red blood cells (RBC) act as a micro bioreactor and protect the enzyme against circulating antibodies. Plasma asparagine diffuses through the RBC membrane to the intra cellular compartment where it is cleaved by the entrapped L-asparaginase. In addition, encapsulation into RBC has been shown to extend the duration of drug action, allowing a reduction in the number of injections.Hepatic toxicity has often been reported with regular L-aparaginase associated with chemotherapy in ALL patients. Therefore this, and more specifically the occurrence of hyperbilirubinemia, with GRASPA, need to be studied. Indeed, bilirubin is a major breakdown product of hemoglobin. Hemoglobin is derived from red cells that have outlived their natural life and subsequently have been trapped inside the spleen. During splenic degradation of RBC, hemoglobin is separated from iron and cell membrane components. Hemoglobin is then transferred to the liver where it undergoes its further metabolic transformations in a process called conjugation. As the liver becomes irritated, the total bilirubin may rise and this lead to hyperbilirubinemia. GRASPA is containing a weak fraction of extracellular hemoglobin (2 g/dl maximum the day of injection), and the red cell half-life has been shown similar to that of non-processed red cells (about 30 days).We investigated in our clinical studies, whether there is any relationship between GRASPA administration and the occurrence of hyperbilirubinemia. In a first study in 12 relapsed-ALL patients <55 years old, at the dosages of 100 and 150IU/kg, we observed 1/6 case of transient hyperbilirubinemia (due to cytolysis) in the adult population and 0/6 case in the children stratum. In the same study, 1/3 adults (due to cytolysis) and 2/3 children, from the control group receiving the native E.Coli L-asparaginase, developped hyperbilirubinemia. In a non-randomised study, 27 newly diagnosed ALL patients over 55 year-old were investigated. At the same dosages, 4/27 experienced hyperbilirubinemia (including 1 with increasing bilirubinemia before treatment). These results are in accordance with data from the literature (W. Stock, 2011, leuk. & lymph.). Interestingly, GRASPA was also administrated in monotherapy in 12 patients with pancreatic carcinoma. Results showed that up to the highest dose tested (150IU/kg) all patients had normal bilirubinemia values.In conclusion, using red cells to vehicle L-asparaginase does not increase the risk of hyperbilirubimenia, and has an acceptable tolerability profile for fragile patients. Disclosures:Plisson:Orphan Europe: Employment. Hunault:ERYTECH Pharma: Principal Investigator Other. Thomas:ERYTECH Pharma: Principal Investigator Other; Orphan Europe: Consultancy. Legay:ERYTECH Pharma: Principal Investigator Other. Bertrand:ERYTECH Pharma: Principal Investigator Other. Andre:ERYTECH Pharma: clinical trial investigator Other. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Renal Anemia Induced by Chronic Ingestion of Depleted Uranium in Rats

Kidney disease is a frequent consequence of heavy metal exposure and renal anemia occurs secondarily to the progression of kidney deterioration into chronic disease. In contrast, little is known about effects on kidney of chronic exposure to low levels of depleted uranium (DU). Study was performed with rats exposed to DU at 40 mg/l by chronic ingestion during 9 months. In the present work, a approximately 20% reduction in red blood cell (RBC) count was observed after DU exposure. Hence, three hypotheses were tested to determinate origin of RBC loss: (1) reduced erythropoiesis, (2) increased RBC degradation, and/or (3) kidney dysfunction. Erythropoiesis was not reduced after exposure to DU as revealed by erythroid progenitors, blood Flt3 ligand and erythropoietin (EPO) blood and kidney levels. Concerning messenger RNA (mRNA) and protein levels of spleen iron recycling markers from RBC degradation (DMT1 [divalent metal transporter 1], iron regulated protein 1, HO1, HO2 [heme oxygenase 1 and 2], cluster of differentiation 36), increase in HO2 and DMT1 mRNA level was induced after chronic exposure to DU. Kidneys of DU-contaminated rats had more frequently high grade tubulo-interstitial and glomerular lesions, accumulated iron more frequently and presented more apoptotic cells. In addition, chronic exposure to DU induced increased gene expression of ceruloplasmin (x12), of DMT1 (x2.5), and decreased mRNA levels of erythropoietin receptor (x0.2). Increased mRNA level of DMT1 was associated to decreased protein level (x0.25). To conclude, a chronic ingestion of DU leads mainly to kidney deterioration that is probably responsible for RBC count decrease in rats. Spleen erythropoiesis and molecules involved in erythrocyte degradation were also modified by chronic DU exposure.