Shortened Red Blood Cells Life Span Research Articles

Complement Activation during Vaso-Occlusive Pain Crisis in Pediatric Sickle Cell Disease

Background: Sickle cell disease (SCD) affects millions of individuals worldwide with substantial morbidity and mortality. The sickle hemoglobin (HbS) polymerizes upon deoxygenation, causing rigid and adhesive red blood cells (RBCs), triggering vascular occlusion, greatly shortened RBC lifespan, and chronic hemolysis. Amongst acute complications in SCD, vaso-occlusive pain crisis (VOC) is the leading cause of hospitalization, with supportive care being the primary approach to management. We and others have recently demonstrated important contributions of complement to the pathophysiology of SCD. When the complement pathway (CP) is activated during SCD crises, inhibition at C5 using eculizumab, has been successful in treating various acute complications in SCD (Chonat et al, Haematologica). In this study, we prospectively analyzed the extent of CP activation among children with SCD presenting with VOC.Methods: Patients aged 0-21 years old managed at Children's Healthcare of Atlanta with homozygous sickle cell (SS) or S beta zero thalassemia genotypes were enrolled in an IRB-approved research study. Inclusion criteria included those requiring intravenous opioids for VOC, and excluded those with chronic pain, >6 VOC admission in the previous 12 months or on chronic transfusions. Blood samples were collected within 48 hours of VOC presentation, and steady-state levels were obtained at a 4-week clinic follow-up. Data was analyzed using a paired t-test, and receiver operator characteristic (ROC) curves were generated comparing intra-person complement levels during acute VOC versus respective steady-state levels.Results: Sixty-four patients have been enrolled thus far, of which 43 (67%) had steady-state samples collected. The majority of patients (90.5%) have SS genotype with a mean (SD) patient age of 14.15 (4.68) years. Fifty-three (84.1%) patients reported taking hydroxyurea (HU). Fifty-nine (93.7%) patients had at least one VOC admission in the past 12 months, with an average of 2.98 (1.67) VOC admissions. Pain Score reported on 55 patients averaged 4.93 (4.78) on a pain scale of 0 to 10. Mean values during VOC and steady-state of hemoglobin (Hb) were 8.12 and 9.01 g/dL, platelet count 431 and 511, and lactate dehydrogenase (LDH) 549 and 483 U/L, respectively. Seventeen patients had complement work-up performed during acute and steady-state, and 4 of them had additional samples collected during subsequent VOC. Complement protein levels C3, C4, C5, properdin, factor B, and complement regulatory proteins factor H and I were unremarkable during VOC and steady-state. However, complement activation markers, specifically anaphylatoxins C3a, C5a and Bb were significantly elevated during VOC compared to steady-state (see Table 1) suggesting activation of alternative CP during VOC (see Table 1 and Figure 1A-C). Terminal complement complex (C5b9) was not statistically different between VOC and steady-state (Figure 1D, red dotted lines signify normal ranges). Remarkably, patients who re-presented with acute VOC exhibited similar increases in their C3a/C5a (Figure 1E-F), substantiating the increases related to their VOC. Hemoglobin and LDH (Figure 1G-H) were similarly significant, suggestive of intravascular hemolysis. Three (7.1%) patients developed acute chest syndrome, two of whom experienced respiratory failure requiring intensive care management, and all exhibited significant CP activation. The area under the curve (AUC) of the ROC curve was analyzed to determine the ability of complement biomarkers to differentiate VOC from steady-state. Based on the AUC of these biomarkers, complement anaphylatoxins C3a and C5a exhibited the highest AUC of 0.76 and 0.87, respectively.Discussion: To our knowledge, this is the first prospective and comprehensive evaluation of CP in patients with SCD during VOC and steady-states. These preliminary findings suggest CP activation is present in a large proportion of patients during VOC, with increased activation of alternative and common CP, associated with intravascular hemolysis. Minimal increase in C5b9 could be explained by a significant proportion (> 80%) of our patients being on HU therapy, similar to prior data (Roumenina et al, AJH). Specifically, C3a/C5a, along with other biomarkers, could not only predict disease activity in patients during VOC, but provide pharmacological targets in VOC, which need further validation. [Display omitted] DisclosuresStowell: Alexion: Consultancy; Argenx: Speakers Bureau; Grifols: Speakers Bureau. Chonat: Alexion: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Takeda: Consultancy, Research Funding.

The Role of Eryptosis in the Pathogenesis of Renal Anemia: Insights From Basic Research and Mathematical Modeling.

Red blood cells (RBC) are the most abundant cells in the blood. Despite powerful defense systems against chemical and mechanical stressors, their life span is limited to about 120 days in healthy humans and further shortened in patients with kidney failure. Changes in the cell membrane potential and cation permeability trigger a cascade of events that lead to exposure of phosphatidylserine on the outer leaflet of the RBC membrane. The translocation of phosphatidylserine is an important step in a process that eventually results in eryptosis, the programmed death of an RBC. The regulation of eryptosis is complex and involves several cellular pathways, such as the regulation of non-selective cation channels. Increased cytosolic calcium concentration results in scramblase and floppase activation, exposing phosphatidylserine on the cell surface, leading to early clearance of RBCs from the circulation by phagocytic cells. While eryptosis is physiologically meaningful to recycle iron and other RBC constituents in healthy subjects, it is augmented under pathological conditions, such as kidney failure. In chronic kidney disease (CKD) patients, the number of eryptotic RBC is significantly increased, resulting in a shortened RBC life span that further compounds renal anemia. In CKD patients, uremic toxins, oxidative stress, hypoxemia, and inflammation contribute to the increased eryptosis rate. Eryptosis may have an impact on renal anemia, and depending on the degree of shortened RBC life span, the administration of erythropoiesis-stimulating agents is often insufficient to attain desired hemoglobin target levels. The goal of this review is to indicate the importance of eryptosis as a process closely related to life span reduction, aggravating renal anemia.

Use of carbon monoxide breath test to assess red blood cell lifespan in newly diagnosed multiple myeloma patients

To clarify the role of red blood cell (RBC) lifespan in anemia of multiple myeloma (MM), RBC lifespan was detected in 40 newly diagnosed MM patients by measuring exhaled endogenous carbon monoxide concentration. Mean RBC lifespan was significantly reduced in MM patients (63 ± 23 d) than healthy controls (116 ± 17 d). RBC lifespan in MM patients without anemia (78 ± 21 d) was also significantly lower than for healthy controls. RBC lifespan in MM patients with anemia (52 ± 18 d) was significantly lower than those without. Besides, RBC lifespan in MM patients with renal insufficiency (50 ± 16 d) was lower than those without (66 ± 23 d). RBC lifespan was significantly negatively correlated with % reticulocytes, erythropoietin concentration, % clonal plasma cells, β2-microglobulin (MG) level, and creatinine level. Receiver operator characteristics curve was used to determine a cut-off point (61 d) to predict effect of RBC lifespan on chemotherapy. Overall response rates were significantly higher in MM patients with RBC lifespan ≥61 d than those <61 d. In conclusion, RBC lifespan of MM patients is reduced, and severely shortened RBC lifespan may be involved in the occurrence of anemia in MM patients. The chemotherapeutic effect of MM patients can be predicted by RBC lifespan.

The life span of red blood cell in patients with severe/very severe aplastic anemia

目的研究重型/极重型再生障碍性贫血（SAA/VSAA）患者的红细胞寿命。方法回顾性分析2016年11月至2017年4月128例SAA/VSAA患者临床资料，以同期13例健康志愿者为正常对照。采用测定内源性一氧化碳呼气试验法检测红细胞寿命，观察SAA/VSAA患者红细胞寿命是否改变及免疫抑制治疗（IST）对红细胞寿命的影响。结果51例初诊未治SAA/VSAA患者红细胞平均寿命为（50.69±21.43）d，较正常对照者[（111.85±31.55）d]明显缩短（t=−6.611，P<0.001）。77例IST组患者红细胞平均寿命为（87.14±39.28）d，其中获得完全治疗反应（CR）、获得治疗反应（HR）、未获治疗反应（NR）患者红细胞平均寿命分别为（106.15±32.12）、（92.00±38.60）、（50.44±21.56）d。获得HR患者红细胞平均寿命较初诊和NR者明显延长（t=7.430，P<0.001；t=4.846，P=0.002），与正常健康对照者接近（t=−1.743，P=0.085）；获得CR患者红细胞平均寿命与正常对照者差异无统计学意义（t=−0.558，P=0.579）。单因素分析未发现影响初诊SAA/VSAA患者红细胞寿命的因素。获得HR的患者与NR患者相比，血清IL-2受体[4.3（1.2~9.7）×105 U/L对6.5（3.7~44.1）×105 U/L，z=−2.733，P=0.006]及IL-6[2.6（2.0~17.7）ng/L对6.1（2.0~14.4）ng/L，z=−2.968，P=0.003]明显减低。初诊组51例患者中38例后续接受IST并评价3个月疗效。采ROC曲线分析治疗前患者红细胞寿命对IST疗效的预测作用，其界值为60 d（敏感性和特异性分别为37.5%和86.4%）。9例治疗前红细胞寿命>60 d患者6例获得HR，29例治疗前红细胞寿命≤60 d患者10例获得HR，差异无统计学意义（P=0.128）。结论SAA/VSAA患者红细胞寿命缩短，IST后可改善或恢复正常。淋巴细胞因子水平升高可能与SAA/VSAA红细胞寿命缩短有关。

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice

AbstractThe factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea–induced mutation that causes a gain of function in adenosine 5′-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection.

Distinct Roles For Hepcidin and Interleukin 6 In The Recovery From Anemia Following Administration Of Heat-Killed Brucella Abortus

Anemia of inflammation (AI) is a complex condition commonly observed in chronic inflammatory states and associated with poor health outcomes and low quality of life. Increased hepcidin (Hamp) expression triggered by interleukin-6 (IL6) is a hallmark of AI and is responsible for iron-restricted erythropoiesis and anemia. Several translational studies have been aimed at neutralizing Hamp overexpression as a therapeutic strategy against AI. However, additional Hamp-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis.In our study, we investigated the distinct roles of iron metabolism and inflammation triggered by Hamp and IL6 in AI. We utilized Hamp-knockout (Hamp-KO), IL6-KO, and wild-type (WT) mice in which we induced inflammation with an intraperitoneal injection of heat-killed Brucella abortus (HKBA). In these mice, we characterized erythropoiesis with weekly CBC, FACS, and red blood cell (RBC) lifespan analysis. In addition, we measured cytokine expression, iron and transferrin (Tf) saturation in sera as indicators of inflammation and iron availability reflecting of iron stores.CBC analysis showed that hemoglobin (Hb) reached a nadir 2 weeks after HKBA injection in WT mice (6.0±1.1 g/dl), and 1 week after HKBA injection in IL6-KO (8.8±1.8 g/dl), and Hamp-KO mice (11.3±1.0 g/dl) (N=10/group), demonstrating that knockout mice were partially protected against HKBA-induced anemia. Additionally, knockout mice fully recovered within 3-4 weeks, whereas WT mice required more than 6 weeks. Mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and reticulocyte hemoglobin content (CHr) were decreased in both WT and IL6-KO mice after HKBA, although values were higher in the latter. Interestingly, Hamp-KO mice showed the highest levels of serum iron, Tf-saturation, MCH, MCV and CHr, indicating that the mild anemia induced by HKBA in these mice was a consequence of superior hemoglobinization of their reticulocytes and RBCs. Iron-depleted Hamp-KO mice were still less sensitive to HKBA administration, suggesting that lack of Hamp expression was contributing to protect these mice from anemia, rather than merely iron overload.FACS analysis performed 3 days after HKBA injection showed that BM erythropoiesis was disrupted in all treated mice. After 1 week, only IL6-KO mice were recovering, although their marrow erythroid progenitors were still reduced compared to PBS-treated mice. The faster recovery in IL6-KO mice was supported by the increased production of reticulocytes compared to Hamp-KO and WT mice. After 1 week, we observed stress erythropoiesis in the spleen of all mice, which was most significant in Hamp-KO, who also showed the highest levels of erythropoiesis in the spleen at steady state. Further investigation of stress erythropoiesis in phlebotomized WT mice injected with HKBA demonstrated that spleen erythropoiesis was partially spared from the inflammatory insult.We showed that a shortened RBC lifespan was contributing to anemia in HKBA-treated mice, and that impaired production of RBCs was accompanied by an increased rate of elimination. In fact, by FACS analysis we characterized macrophages (Gr1-/CD115-/SSClow/F4/80hi) engulfing erythroid cells (Ter119+) by discriminating multiplets vs. single cells (SSC-H vs. SSC-A), and observed that hemophagocytosis was occurring in HKBA-treated mice.Finally, we analyzed 58 inflammatory cytokines (RodentMap, Myriad RBM) from mouse sera and showed that HKBA administration changed the level of expression of inflammatory molecules more substantially in Hamp-KO than in IL6-KO compared to WT mice (26 cytokines changed in Hamp-KO mice, with fold change > or <1.5 vs. 1 changed in IL6-KO mice; P<.001). This indicated that lack of Hamp influences cytokine expression more significantly than lack of IL6 after HKBA injection. Further analyses are in progress to define the function and pathways that correlate the different cytokines.In conclusion, we showed that both IL6 and Hamp ablation offered protection against AI and that IL6-KO and Hamp-KO mice exhibited different patterns in the development and resolution of anemia. We believe that our data support the notion that not only IL6 and Hamp play distinct roles in modulating erythropoiesis in AI, but also that cytokines could be targeted as a therapeutic approach to improve marrow erythropoiesis in AI.</abstract> Disclosures:Sasu:Amgen Inc.: Employment. Cooke:Amgen Inc.: Employment. Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

A new paradigm in the diagnosis of hereditary hemolytic anemia

A new paradigm in the diagnosis of hereditary hemolytic anemia

Aminophospholipid Translocase Activity and Phosphatidylserine Externalization in Sickle Red Blood Cell Subpopulations.

Red blood cell (RBC) membrane phosphatidylserine (PS) is normally confined to the inner leaflet. In sickle RBC, however, PS externalization has been observed and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Increased calcium leads to PS externalization through inhibition of aminophospholipid translocase (APLT), which normally returns external PS to the inner leaflet, and activation of phospholipid scramblase, which allows nonspecific phospholipid equilibration between the leaflets. Sickle RBC in the light and dense (dehydrated) fractions exhibit increased PS externalization compared to sickle RBC in the normal density fractions. Sickle RBC with modest PS exposure (Type I PS+) occur in both light and dense fractions and, especially in the light fractions, include many reticulocytes. Sickle Cells with high levels of PS exposure (Type II PS+) occur predominantly in the dense fraction. Type II PS+ sickle cells have low levels of HbF, are more adherent than Type I PS+ cells, and tend to increase in number as sickle cells age in the circulation. Dense sickle cells have decreased APLT activity and this has been considered necessary (but probably not sufficient) for PS externalization. In the current studies we have examined the relationship between PS externalization and translocase activity in sickle cell density fractions. APLT activity and PS externalization were determined flow cytometrically using NBD-PS internalization and annexin V-phycoerythrin binding respectively. Sickle RBC density fractions were isolated using a discontinuous Optiprep® gradient, and two fractions were selected for further study, Fx 4 with essentially normal hydration (1.093< g/cc <1.100) and the dehydrated Fx 6 (> 1.120 g/cc). As expected, Fx 6 sickle RBC had a decreased percentage of APLT positive cells (29.9±10.8, N=4) compared to normal RBC (94.5±3.6, N=3) and Fx 4 sickle RBC (92.9±2.3, N=4). Fx 4 contained 5.7±2.8% Type I and 0.3±0.2% Type II PS+ cells. Fx 6 had 9.1±2.5% Type I and 3.2±2.3% Type II PS+ cells. Figure 1 shows the percentage of APLT positive cells in the PS negative (PS-), Type I PS+, and Type II PS+ groups for Fx 4 and Fx 6. Type I PS+ cells in Fx 4 are positive for APLT, whereas those in Fx 6 are mostly negative. In Fx 4, the Type I PS+ group most likely contains many reticulocytes, and in a separate experiment TfR+ reticulocytes had a uniform, slightly elevated APLT activity. The Type II PS+ cells in both Fx 4 and Fx 6 had decreased percentages of APLT positive cells. It thus appears that high levels of PS externalization, regardless of hydration state, are associated with low APLT activity. We therefore conclude:In normally hydrated sickle cells, Type II PS+ cells, but not Type I PS+ cells, are associated with decreased APLT;In dense sickle RBC, both Type I and Type II PS+ cells are associated with decreased APLT. These results further emphasize the pathologic nature of the Type II PS+ sickle cells. [Display omitted]

Simplification of the end-alveolar carbon monoxide technique to assess erythrocyte survival

We have described a simple, rapid method for determining red blood cell (RBC) life span based on measurement of alveolar carbon monoxide (CO) concentration corrected for atmospheric CO as determined with a device that simulates the body’s equilibration with CO. This study was designed to determine whether direct measurements of ambient CO can be substituted for sampling with the equilibrator device. Atmospheric samplings obtained in our medical center over a period of weeks indicated that CO concentration was sufficiently constant that a single ambient CO measurement at the time of breath collection adequately corrected for atmospheric CO. Additional studies with volunteers showed that overnight CO concentrations in the subjects’ home environments also remained relatively constant, suggesting that accurate assessment of RBC life span can be obtained from alveolar CO concentrations and home atmospheric samples collected on a subject’s arising in the morning. Using this technique, we found that the RBC life span of 40 healthy volunteers averaged 122 ± 23 days, a value comparable to that obtained with complex cohort labeling methods. No significant correlation was observed between RBC life span and reticulocyte count in these individuals. The ability of this simple technique to detect increased RBC turnover was demonstrated in 4 subjects being treated with ribavirin and interferon for hepatitis C, a treatment reported to shorten RBC life span. Measurement of CO in samples collected by these four subjects in their home environments indicated that each had a shortened RBC life span (range 30–69 days).

Physiological significance and molecular genetics of red cell enzymes involved in the ribonucleotide metabolism

At the final maturation process red blood cells (RBC) are enucleated, becoming unable to synthesize nucleic acids as well as proteins. RBCs survive approximately 120 days in circulation using glucose as the sole energy source. Most crucial RBC functions depend on ATP to sustain physiological homeostasis. It is thus quite important that generation of ATP by glycolysis and replenishing of adenine nucleotide pools by the reaction, which is catalyzed by adenylate kinase (AK1). In turn, ribosomal RNA is degraded during remodeling of reticulocytes, and pyrimidine ribonucleotides become unnecessary for RBC viability. Thus they should be dephosphorylated by pyrimidine 5'-nucleotidase (P5N-I) and finally transported outside RBCs. There have been reported that hereditary deficiency of AK1 and P5N-I may cause shortened RBC life span, i.e. hemolytic anemia. In this review, we summarize physiological importance of these enzymes, which are involved in ribonucleotides metabolism during RBC maturation.

198 RED BLOOD CELLS IN PRETERM AND TERM NEONATES SHOW DECREASED RESISTANCE TO PLASTIC DEFORMATION WHEN COMPARED TO ADULTS

The mechanical fragility of red blood cells (RBC) in term neonates is increased compared with adult RBC. We studied tether formation (i.e., small area membrane rupture) of single RBC from preterm infants, term neonates and adults using a new micropipette technique. Point attached RBC were aspired at different negative pressures into a large micropipette with an internal diameter of about 7.8 μm.We measured (1) the critical presure to start membrane tether formation, (2) the onset of tether formation, (3) the lenth of membrane tethers after 2, 5, 10, 15, 20, 25, 30 s and (4) the relaxation behavior of the membrane tethers after releasing the RBC. At a pressure of −3mm H2O. Only RBC of preterm infants began to form tethers earlier (3.7s) than term neonates (8.2s). The tether elongation rate was also increased in preterm infants. At a pressure of −5mm H2O, We observed also tether formatoin in adult RBC. Tether formation at a pressure of −5mm H2O began after 10.2 s in adults, 4.1 s in term neonats and 3.2 s in preterm infants. 10 s after the begining of tether formation, the tether lengts were 22.15 ± 8.05 μm in preterm neonats, 11.8 ± 3.62 μm in term neonats, and 10.63 ± 4.97 μm in adults. The relaxation behavior of the tethers was similar in the three groups. Repeated tether formation of the same poi attached RBC changed the pattern of tether formation. This indicates irreversible of the cell membrane during tether formation. We conclude, the RBC in preterm infants show decreased mechanical resistance compared with full term neonats. Decreased resistance of neonatal RBC to tether formation may contribute to the shortened RBC life span, particularly in preterm infants.

Intravascular hemolytic syndrome following aortic valve replacement. Complete hematologic and cardiac recovery with surgical intervention

INTRAVASCULAR hemolytic anemia and bizarre red blood cell fragmentation has been an infrequent but spectacular complication of open heart surgery. More specifically, this syndrome has occurred in association with defective intracardiac prosthetic implants, both interatrial patch grafts^1-7and valve prostheses.7-16 Of the 15 patients with well-documented "traumatic" hemolytic anemia following aortic valve replacement, only two instances of complete hematologic recovery have been noted, both of which attended replacement of the defective prosthesis.⁸Excluding five deaths, the remaining eight patients all showed evidence of persistent hemolytic anemia or a compensated hemolytic state, ie, reticulocytosis, red blood cell (RBC) fragmentation, and a shortened RBC life span (Table). We have recently studied a patient who developed intravascular hemolytic anemia following implantation of an aortic cagedball valve prosthesis. Although the hematologic features of his illness were similar to previously reported cases, the ultimate course of the hemolytic process was unique in that

Intravascular Hemolytic Syndrome Following Aortic Valve Replacement

INTRAVASCULAR hemolytic anemia and bizarre red blood cell fragmentation has been an infrequent but spectacular complication of open heart surgery. More specifically, this syndrome has occurred in association with defective intracardiac prosthetic implants, both interatrial patch grafts1-7and valve prostheses.7-16 Of the 15 patients with well-documented traumatic hemolytic anemia following aortic valve replacement, only two instances of complete hematologic recovery have been noted, both of which attended replacement of the defective prosthesis.8Excluding five deaths, the remaining eight patients all showed evidence of persistent hemolytic anemia or a compensated hemolytic state, ie, reticulocytosis, red blood cell (RBC) fragmentation, and a shortened RBC life span (Table). We have recently studied a patient who developed intravascular hemolytic anemia following implantation of an aortic cagedball valve prosthesis. Although the hematologic features of his illness were similar to previously reported cases, the ultimate course of the hemolytic process was unique in that