Red cell exchange employing phenotypically matched deglycerolized red blood cells to treat acute sickle cell crisis: A case report

2009 ◽  
Vol 41 (2) ◽  
pp. 155-156
Author(s):  
Cesare Perotti ◽  
Paola Isernia ◽  
Claudia Del Fante ◽  
Gianluca Viarengo ◽  
Laura Salvaneschi ◽  
...  
Keyword(s):  
Case Report ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Red Cell ◽  
Sickle Cell Crisis

scholarly journals Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-30
Author(s):  
Yuanbin Song ◽  
Rana Gbyli ◽  
Liang Shan ◽  
Wei Liu ◽  
Yimeng Gao ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Red Cell ◽  
Cell Disease ◽  
Ineffective Erythropoiesis ◽  
Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1577-1584 ◽  
Author(s):  
Kitty de Jong ◽  
Renee K. Emerson ◽  
James Butler ◽  
Jacob Bastacky ◽  
Narla Mohandas ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Cell Model ◽  
Red Cell ◽  
Sickle Cells ◽  
Red Cell Survival ◽  
Transgenic Murine Models

Several transgenic murine models for sickle cell anemia have been developed that closely reproduce the biochemical and physiological disorders in the human disease. A comprehensive characterization is described of hematologic parameters of mature red blood cells, reticulocytes, and red cell precursors in the bone marrow and spleen of a murine sickle cell model in which erythroid cells expressed exclusively human α, γ, and βS globin. Red cell survival was dramatically decreased in these anemic animals, partially compensated by considerable enhancement in erythropoietic activity. As in humans, these murine sickle cells contain a subpopulation of phosphatidylserine-exposing cells that may play a role in their premature removal. Continuous in vivo generation of this phosphatidylserine-exposing subset may have a significant impact on the pathophysiology of sickle cell disease.

scholarly journals High-Throughput Mirna Analysis Suggests Pro-Inflammatory Profile in Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1077-1077
Author(s):  
Matthew Cannon ◽  
Sarah Glass ◽  
Sidney Smith ◽  
Melanie Heinlein ◽  
Rosa Lapalombella ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Research Funding ◽  
Red Cell ◽  
Cell Disease ◽  
Acute Crisis

Abstract BACKGROUND: Mature circulating red blood cells, though devoid of a nucleus, have been shown to contain an abundance of miRNAs. Further, it has been shown that sickle cell patient-derived RBCs have a dramatic difference in miRNA content than normal RBCs. Given that a range of miRNAs are involved in the regulation of immunity, including the release of inflammatory mediators, we hypothesize that miRNAs enriched in circulating red blood cells function to prolong the inflammatory state in sickle cell disease. Further, we hypothesize that these miRNAs can be used as biomarkers for use in the clinic to predict crisis and differentiate acute versus chronic pain. Exploring this miRNA enrichment in circulating red blood cells in sickle cell patients will provide practical insight for the inflammation state and will inform characteristics of patients who may need greater care in the clinic. METHODS: Twenty steady state patients were recruited and categorized according to their chronic pain status and crisis frequency per year. Whole blood was drawn during routine visits to the OSU Wexner Medical Center Hematology Clinic. Additionally, whole blood was drawn from five patients either in acute pain crisis (recruited prior to crisis) or within a few days of crisis. Samples were subject to double gradient centrifugation and red cells were resuspended in Trizol and cryopreserved. MiRNAs were isolated from red cell Trizol suspensions using a commercial isolation kit (QIAGEN Cat#217004). Isolated miRNAs were then subject to a NanoString Human miR (v3) expression assay. Differential expression analysis was conducted to compare miRNAs with at least 1.5 fold difference (p = 0.05) between steady state and acute crisis. Target prediction and GO ontology analysis was performed for statistically significant miRNAs using DIANA Tools mirPath v3. Follow-up qPCRs were performed using TaqMan Advanced miRNA cDNA Synthesis Kit (Cat#A28007) and TaqMan Advanced miRNA Assays (Cat#A25576) to validate the decreased expression of miRNAs. Additional qPCRs were performed using TaqMan Gene Expression Assays (Cat#4331182) to investigate mRNA regulatory effects of significant miRNAs in the total red cell population. Western blots were also performed to investigate regulatory effects of these miRNAs at the protein level. RESULTS & CONCLUSION: Comparison of RBC miRNA profiles from patients during acute crisis to those in steady state shows several significantly decreased (>1.5 fold) miRNAs in crisis. Among these miRs we have found previously uncharacterized miRNAs, hsa-miR-2116-5p and hsa-miR-302d-3p. DIANA tools miRNA analysis software predicts these miRNAs to be involved in regulation of cell-to-cell adhesion pathways through gene transcripts such as Protocadherin Beta 6 (PCDHB6) and Neural Cell Adhesion Molecule 2 (NCAM2). Interestingly, inspection of miRNA predicted targets that fall under significant GO terms also predicts several individual miRNAs to regulate inflammatory response and nociceptive signaling gene transcripts like A20 (TNFAIP3) and Cathepsin S (CTSS). Validation of these miRNAs was performed via qPCR for 5 out of the 6 significantly decreased miRNAs. Of the 5 miRNAs tested, hsa-miR-2116-5p, hsa-miR-302d-3p, and hsa-miR-1246 were validated as having decreased expression in acute crisis patients compared to steady state. qPCRs were then performed to probe for miRNA based regulation of top predicted target mRNA transcripts. Both CTSS and TNFAIP3 showed increased expression of mRNA transcripts in acute crisis patient red cells as compared to steady state. Next, western blot analysis was performed on red cell protein lysate. Interestingly, this analysis revealed a pattern in activated CTSS expression that was independent of acute crisis. Steady state patients reporting chronic pain showed increased activated CTSS compared to those without chronic pain. Activated CTSS was not found in red cell lysates from three normal, non-SCD donors. Taken together, these results suggest that red blood cells may play a larger role in inflammation and pain responses in sickle cell disease than previously thought. Further these results suggest activated CTSS as a potential biomarker for differentiating chronic pain in patients. Follow-up studies are underway to further stratify and investigate these findings. Disclosures Desai: University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Committee; NIH: Research Funding; FDA: Research Funding; Selexy/Novartis: Research Funding; Pfizer: Research Funding.

scholarly journals Red Cell Rheology Biomarkers to Assess Cure in Gene-Based Therapies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Celeste K. Kanne ◽  
Danielle Guffey ◽  
Charles Minard ◽  
Eduard van Beers ◽  
Richard van Wijk ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Research Funding ◽  
Room Temperature ◽  
Cell Transplant ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Oxygen Gradient ◽  
Cell Rheology

Introduction Red cell rheology is abnormal in sickle cell disease (SCD); red blood cells (RBC) are rigid, dense, and the sickle hemoglobin (HbS) polymerizes with deoxygenation. There are several devices commercially available and under development to assess RBC rheology. One is an oxygen gradient ektacytometer (Lorrca with Oxygenscan, RR Mechatronics) which measures RBC deformability under oxygenated conditions (EImax) and deoxygenated conditions (EImin), and the oxygen concentration at which deformability begins to worsen, point of sickling (PoS). A commercially available hematology analyzer, the ADVIA (Siemens), measures hemoglobin (Hb) by flow and colorimetric methods, permitting automated calculation of the percent dense red blood cells (%DRBC). Allogeneic hematopoietic stem cell transplant (alloHSCT) can provide a cure for SCD, and viable gene-based therapy options are under investigation to serve the many patients without a matched related donor. However, the level of HbS correction or functional Hb induction necessary to achieve a cure is still unknown. Clinical endpoints such as prevention of pain events are important, but it is possible to be pain free for several years while still experiencing organ damage. As gene-based therapy clinical trials move forward, we must assess the level of functional improvement beyond Hb profile and conventional clinical labs. We propose that the goal of any gene-based SCD therapy should be to normalize blood rheology to the level of an individual with sickle cell trait (HbAS), and that EImax, EImin, PoS, and %DRBC may be used to distinguish between HbAS and HbSS/Sβ0 genotypes. Methods Subjects: Blood samples were collected from 257 unique patients (17 HbAS, and 240 HbSS/Sβ0) under IRB-approved protocols at Texas Children's Hospital and University Medical Center Utrecht. Patients were 56% male, ages 9 months to 22 years. Some HbSS/Sβ0 subjects were on transfusion and hydroxyurea (HU) (Table 1). Fetal hemoglobin (HbF) levels ranged from 0-41%. Oxygen gradient ektacytometry: Blood collected in EDTA and standardized to a fixed RBC count was suspended in 5 mL polyvinylpyrrolidone at room temperature. 1.5 mL of the sample solution was injected into test cup. Each sample was run in duplicate. ADVIA: 250µL of blood collected in EDTA at room temperature was aspirated to measure the %DRBC, defined as the percentage of RBCs with a Hb concentration >1.11 mg/mL. Analysis: Patient characteristics were summarized using median with 25th and 75th percentiles, and frequency with percentage. Characteristics and labs were compared by group with t-test, Wilcoxon rank sum test, or Fisher's exact test. Receiver operating characteristics (ROC) analyses were performed to identify HbAS versus HbSS/Sβ0 for each biomarker. All analyses were performed using Stata 15. Results The EImin, EImax, PoS, and %DRBC differed significantly between the HbAS and HbSS/Sβ0 groups, despite including transfused, very young, and HU-treated samples in the HbSS/Sβ0 cohort (p<0.001, p=0.002, p<0.001, and p<0.001, respectively; Table 1). ROC analysis showed that a threshold Elmin greater than or equal to 0.453 identified HbAS with 94.12% sensitivity and 95.43% specificity. EImax greater or equal to 0.569 identified HbAS with 94.12% sensitivity and 81.57% specificity. PoS less than 21.38 mmHg identified HbAS with 93.87% sensitivity and 93.33% specificity. %DRBC less than 1.6% identified HbAS with 88.13% sensitivity and 88.00% specificity (Table 2). Conclusion Conventional laboratory testing may not be able to establish if gene-based therapy has achieved a cure. We propose to define a cure as achievement of an HbAS level of RBC quality in a gene-based therapy edited RBC population. We show that rheological biomarkers EImin, EImax, PoS, and %DRBC differ significantly between individuals with HbAS and HbSS/Sβ0 of all spectrums of severity and treatment regimens. Not only are the rheological biomarker values significantly different, but there is little to no overlap in ranges of values obtained from the different genotypes, even in heavily transfused HbSS patients, with HbS as low as 31%. EImin, EImax, PoS, and %DRBC functionally identify genotype with high sensitivity and specificity; we propose that they be used not to diagnose SCD, but to determine if a HbSS or HbSS/Sβ0 individual who has undergone successful gene-based therapy has achieved the RBC functionality of a HbAS individual. Disclosures van Beers: Novartis: Research Funding; Pfizer: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR mechatronics: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Rab:RR Mechatronics: Research Funding. Sheehan:Global Blood Therapeutics: Research Funding; Novartis: Research Funding; Emmaus: Research Funding.

Ion Transport Pathology in the Mechanism of Sickle Cell Dehydration

2005 ◽  
Vol 85 (1) ◽  
pp. 179-200 ◽  
Author(s):  
Virgilio L. Lew ◽  
Robert M. Bookchin
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Experimental Tests ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Open Questions ◽  
Gardos Channel ◽  
Cell Dehydration ◽  
Experimental Findings

Polymers of deoxyhemoglobin S deform sickle cell anemia red blood cells into sickle shapes, leading to the formation of dense, dehydrated red blood cells with a markedly shortened life-span. Nearly four decades of intense research in many laboratories has led to a mechanistic understanding of the complex events leading from sickling-induced permeabilization of the red cell membrane to small cations, to the generation of the heterogeneity of age and hydration condition of circulating sickle cells. This review follows chronologically the major experimental findings and the evolution of guiding ideas for research in this field. Predictions derived from mathematical models of red cell and reticulocyte homeostasis led to the formulation of an alternative to prevailing gradualist views: a multitrack dehydration model based on interactive influences between the red cell anion exchanger and two K+transporters, the Gardos channel (hSK4, hIK1) and the K-Cl cotransporter (KCC), with differential effects dependent on red cell age and variability of KCC expression among reticulocytes. The experimental tests of the model predictions and the amply supportive results are discussed. The review concludes with a brief survey of the therapeutic strategies aimed at preventing sickle cell dehydration and with an analysis of the main open questions in the field.

scholarly journals Increased IgG molecules bound to the surface of red blood cells of patients with sickle cell anemia

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 301-304 ◽  
Author(s):  
LD Petz ◽  
P Yam ◽  
L Wilkinson ◽  
G Garratty ◽  
B Lubin ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Red Cells ◽  
Antibody Activity ◽  
Red Cell ◽  
Indirect Antiglobulin Test ◽  
Sickle Cell Crises ◽  
Transfusion History

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.

scholarly journals Increased IgG molecules bound to the surface of red blood cells of patients with sickle cell anemia

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 301-304 ◽  
Author(s):  
LD Petz ◽  
P Yam ◽  
L Wilkinson ◽  
G Garratty ◽  
B Lubin ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Red Cells ◽  
Antibody Activity ◽  
Red Cell ◽  
Indirect Antiglobulin Test ◽  
Sickle Cell Crises ◽  
Transfusion History

Abstract We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.

Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

1996 ◽  
Vol 76 (03) ◽  
pp. 322-327 ◽  
Author(s):  
Dominique Helley ◽  
Amiram Eldor ◽  
Robert Girot ◽  
Rolande Ducrocq ◽  
Marie-Claude Guillin ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Annexin V ◽  
Mean Value ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Cell Disease ◽  
Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

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