Purification Platelets from Mouse Blood with Sickle Cell Disease Using Iohexol Gradient Medium

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. Since RBC and WBC contain significantly more RNA and proteins than platelets, the presence of even a small number of these cells can interfere with transcriptomic and proteomic analyses of RNA and proteins derived from platelets. Several protocols have described the isolation of platelets from human, dog, rat, and non-human primate by various methods. Some of the methods require multiple steps such as collection of platelet-rich plasma by centrifugation, filtration by separation column, negative selection of platelets with RBC- and WBC-specific antibody conjugated to magnetic beads, and so on, which are time-consuming and may degrade platelets and their contents. Moreover, mouse blood with sickle cell disease contains a significant level of fragments of RBC which should be removed from the platelet preparation. However, the mouse yields a relatively smaller volume of blood, which makes it difficult to purify platelets. If the same small volume of gradient medium and blood samples are used, the platelet layer cannot be clearly separated from RBC-WBC layer after centrifugation. We describe a simple method for purification of platelets from mouse blood with sickle cell disease using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The platelet layer is collected and centrifuged again at 200 x g for 20 min at 20 °C to remove the residual fragments of RBC. The recovery/yield of the purified platelets were 10-17%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets were activated with thrombin indicating their viability. We confirmed that the purified platelets were sufficiently pure using flow cytometric and microscopic evaluation. Although flow cytometric analysis of purified platelet from sickle cell disease mice showed a few RBC events after staining with anti-TER119 antibody, the microscopic study did not show any intact RBC or larger fragments indicating that these are smaller fragments of RBC which do not interfere with the biochemical and functional studies. This method can be used for purification of platelets from the blood of other species and disease models as well. Disclosures No relevant conflicts of interest to declare.

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Increased Expression of Type 1 Interferon Stimulated Genes in Sickle Cell Disease and a Potential Association with RBC Alloimmunization

Blood ◽

10.1182/blood-2019-124899 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 716-716

Author(s):

Emaan Madany ◽

Najwa El Kadi ◽

Sumaarg Pandya ◽

Jeanne E. Hendrickson ◽

David R Gibb

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Flow Cytometric Analysis ◽

Gene Signature ◽

Cell Disease ◽

Interferon Stimulated Genes ◽

Flow Cytometric ◽

Increased Risk ◽

Significant Difference

Background RBC transfusion can lead to the production of alloantibodies against RBC antigens and is a clinically significant issue in transfusion medicine. Patients with sickle cell disease have an increased risk for alloimmunization; 30-50% of SS patients have alloantibodies compared to 3-10% of other hospitalized patients. These alloantibodies can cause dangerous hemolytic transfusion reactions and limit the availability of compatible antigen-negative RBC products. This is of particular importance in SS patients, who commonly make alloantibodies against multiple RBC antigens and need regular transfusions to treat their disease. However, mechanisms underlying the increased frequency of alloimmunization in sickle cell patients are poorly understood. In previous studies, inflammation in the recipient has been shown to promote alloimmunization in both mice and humans. In mouse transfusion models, type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) have been shown to promote alloimmunization. Other studies have shown that patients with inflammatory autoimmune diseases express an IFNα/β signature, which may contribute to the increased frequency of alloimmunization in these populations. Given the chronic inflammatory state in patients with sickle cell disease, we hypothesize that: SS patients may also have an IFN gene signature that may contribute to the increased frequency of alloimmunization. Methods To test this hypothesis, we initially measured the expression of the ISG, myxovirus resistance protein 1 (MxA), in the blood of previously-transfused patients (n=50) with SS disease (SS, n=13) and without SS disease (ββ, n=37) by whole blood immunoassay (ELISA). We then measured expression of another ISG, Siglec-1 (SS n=5, ββ=24), expressed on monocytes by flow cytometric analysis. To determine the degree to which ISG expression correlated with alloimmunization frequency, expression of MxA in non-alloimmunized patients was compared to the expression in patients with 1 or more alloantibodies. Statistical analysis of 2 groups was completed with a Mann-Whitney U test. Significance between 3 groups was determined using a Kruskal-Wallis test with a Dunn's post-test. Results SS patients had significantly elevated levels of MxA (mean ± standard error of the mean, SS MxA = 8.98 ng/mL ± 2.46) compared to control patients without SS (MxA = 1.25 ± 0.54, p<0.0001). (Figure 1 A). SS patients also had significantly elevated levels of Siglec-1 on blood monocytes, measured by flow cytometric mean fluorescence intensity (MFI, SS MFI = 132.72 ± 42.9, ββ MFI = 64.9 ± 6.17, p< 0.05). (Figure 1 B,C). For all 50 patients, including SS and ββ control patients, there was a trend toward elevated MxA expression in alloimmunized patients. Patients with 2 or more alloantibodies had significantly elevated MxA (MxA 8.16 ± 2.61), compared to non-alloimmunized transfused patients (MxA = 2.05 ± 1.65, p < 0.01) or patients with only 1 alloantibody (MxA = 1.18 ±0.48, p<0.01). There was no significant difference in MxA levels between patients with 0 and 1 alloantibody. Of the 13 patients with SS disease, only 2 patients lacked alloantibodies. (SS with 1 alloantibody, n=3, SS with 2 or more alloantibodies, n=8). Therefore, a correlation between MxA levels and alloimmunization in SS patients could not be assessed. Discussion Factors that contribute to RBC alloimmunization in sickle cell disease are poorly understood. In this study, we found that sickle cell patients had an increase in the expression of ISGs compared to other transfused patients. We also found that MxA levels are increased in patients that have 2 or more alloantibodies compared to patients without alloantibodies. These findings suggest the presence of an IFNα/β gene signature in patients with sickle cell disease. Further studies are needed to determine the relationship between interferon-stimulated responses in sickle cell patients and the increased frequency of alloantibody production. Disclosures No relevant conflicts of interest to declare.

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Expression Pattern of CD55 and CD59 on Red Blood Cells in Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.4815.4815 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4815-4815

Author(s):

Salah A. Al Humood ◽

Lama A. Al-Faris ◽

Monera Al-Rukhayes

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Healthy Controls ◽

Cell Disease ◽

Altered Expression ◽

Cd59 Expression

Abstract Background: Altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, might result in increased susceptibility of red blood cells (RBCs) to complement-mediated lysis. Limited information is available on the pattern of CD55 and CD59 expression on RBCs of sickle cell disease (SCD) patients. Methods: Flow cytometric analysis was performed on RBCs from 71 adult SCD patients and 53 healthy controls, using the commercial REDQUANT kit. Results: CD59 deficiency was significantly higher among SCD patients than among healthy controls. The proportions of CD55-deficient and CD59-deficient RBCs from SCD patients were significantly higher when compared with those from healthy controls (0.17 vs. 0.09 and 2.1 vs. 1.2, respectively). The MFI of CD55 and CD59 expression on RBCs in SCD was significantly reduced when compared to the expression healthy controls (5.2 vs. 6.4 and 19.4 vs 20.3, respectively). The pattern of CD55 and CD59 expression was not correlated with anemia, biomarkers of hemolysis, erythropoietin level or other pro-inflammatory markers. Conclusions: There is an altered pattern of CD55 and CD59 expression on RBCs of SCD Patients; however, it does not seem to play a causal role in the pathophysiology of anemia, and is unlikely to be influenced by the level of erythropoietin or other inflammatory mediators. Disclosures No relevant conflicts of interest to declare.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650577 ◽

1996 ◽

Vol 76 (03) ◽

pp. 322-327 ◽

Cited By ~ 73

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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Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽

10.1039/d0lc01133a ◽

2021 ◽

Author(s):

Yuncheng Man ◽

Debnath Maji ◽

Ran An ◽

Sanjay Ahuja ◽

Jane A Little ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Red Blood Cells ◽

Sickle Cell ◽

Red Blood Cell ◽

Blood Cells ◽

Electrical Impedance ◽

Cell Disease ◽

Blood Disorders

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contributes to vaso-occlusion and disease pathophysiology. However, there are few...

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Role of Adhesion Molecules and Vascular Endothelium in the Pathogenesis of Sickle Cell Disease

Hematology ◽

10.1182/asheducation-2007.1.84 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 84-90 ◽

Cited By ~ 27

Author(s):

Marilyn J. Telen

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Adhesion Molecules ◽

Sickle Cell ◽

Vascular Endothelium ◽

Blood Cells ◽

Cell Disease ◽

Adhesive Interactions ◽

Lines Of Evidence

AbstractA number of lines of evidence now support the hypothesis that vaso-occlusion and several of the sequelae of sickle cell disease (SCD) arise, at least in part, from adhesive interactions of sickle red blood cells, leukocytes, and the endothelium. Both experimental and genetic evidence provide support for the importance of these interactions. It is likely that future therapies for SCD might target one or more of these interactions.

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Influence of Haptoglobin Polymorphism on Stroke in Sickle Cell Disease Patients

Genes ◽

10.3390/genes13010144 ◽

2022 ◽

Vol 13 (1) ◽

pp. 144

Author(s):

Olivia Edwards ◽

Alicia Burris ◽

Josh Lua ◽

Diana J. Wilkie ◽

Miriam O. Ezenwa ◽

...

Keyword(s):

Oxidative Stress ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Cells ◽

Cell Disease ◽

Free Hemoglobin ◽

Cerebral Tissue ◽

Sickle Hemoglobin ◽

Haptoglobin Polymorphism

This review outlines the current clinical research investigating how the haptoglobin (Hp) genetic polymorphism and stroke occurrence are implicated in sickle cell disease (SCD) pathophysiology. Hp is a blood serum glycoprotein responsible for binding and removing toxic free hemoglobin from the vasculature. The role of Hp in patients with SCD is critical in combating blood toxicity, inflammation, oxidative stress, and even stroke. Ischemic stroke occurs when a blocked vessel decreases oxygen delivery in the blood to cerebral tissue and is commonly associated with SCD. Due to the malformed red blood cells of sickle hemoglobin S, blockage of blood flow is much more prevalent in patients with SCD. This review is the first to evaluate the role of the Hp polymorphism in the incidence of stroke in patients with SCD. Overall, the data compiled in this review suggest that further studies should be conducted to reveal and evaluate potential clinical advancements for gene therapy and Hp infusions.

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

Blood ◽

10.1182/blood-2020-141603 ◽

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.

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The prevalence of leukocyte abnormalities among Sudanese patients with sickle cell disease

World Journal of Advanced Research and Reviews ◽

10.30574/wjarr.2021.9.1.0026 ◽

2021 ◽

Vol 9 (1) ◽

pp. 262-267

Author(s):

Tarig Osman Khalafallah Ahmed ◽

Ekhlas Alrasheid Abu Elfadul ◽

Ahmed A. Agab Eldour ◽

Omer Ibrahim Abdallah Mohammed

Keyword(s):

Sickle Cell Disease ◽

Cell Volume ◽

Sickle Cell ◽

Blood Cells ◽

White Blood Cells ◽

Blood Film ◽

Cell Disease ◽

Mean Cell Volume ◽

Blood Disorder ◽

Age Range

Sickle cell disease (SCD) is an inherited blood disorder that affects red blood cells. The study was conducted in Elobied town during the period May 2011 to September 2011. The aim of this study is to detect the abnormalities of leucocytes among sickle cell anemic patients. 40 sickle cell anemic patients; age range between 8 months to 23 years. Blood sample was taken for all patients and the laboratory investigation were performed using automated estimation for: hemoglobin (Hb), Packed cell volume (PCV), red cell count (RBCs), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell concentration (MCHC), and total white blood cells, comment on blood film using manual methods. The conclusion of this study there is increase in total white blood cells with shift to left in neutrophil precursor in sickle cell patients with complications ,the most immature cells are band form, myelocytes and metamyelocytes, and there also lymphocytosis and neutrophilia which has been increases in response to infections.

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