Measuring sickle cell morphology in flow using spectrally encoded flow cytometry (Conference Presentation)

2017 ◽  
Author(s):  
Inna Kviatkovsky ◽  
Adel Zeidan ◽  
Daniella Yeheskely-Hayon ◽  
Eldad J. Dann ◽  
Dvir Yelin
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell ◽  
Cell Morphology ◽  
Spectrally Encoded

Evaluation of Cell Types and Morphologies in Sickle Cell Disease with an Imaging Flow Cytometer

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 972-972 ◽  
Author(s):  
Hasan Tahsin Ozpolat ◽  
Tim Chang ◽  
Junmei Chen ◽  
Xiaoping Wu ◽  
Colette Norby ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell ◽  
Cell Morphology ◽  
Research Funding ◽  
Hemoglobin Concentration ◽  
Cell Types ◽  
Blood Film ◽  
Hypotonic Solution ◽  
Dense Cell ◽  
Activation Status

Abstract Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusive episodes and hemolysis. Hemoglobin S is prone to polymerize at low oxygen tension, causing the red cell to become sickle shaped, more rigid and sticky. Evaluation of blood cell morphology, counts and activation are important components of the patient evaluation. This is usually accomplished by evaluation of the blood film, performing a complete blood count (CBC), and with the use of flow cytometry. A typical blood film from an SCD patient shows anisocytosis, poikilocytosis, polychromasia, nucleated erythrocytes, sickled cells, and irregular contracted cells. The methods of blood cell evaluation all have disadvantages. Preparation of the blood film is laborious and its evaluation is highly subjective and requires extensive experience. Some CBC counters (e.g., Siemens - ADVIA 2120) are able to detect dense cells (increased hemoglobin content-high MCHC cells) by their volume and hemoglobin concentration after the red blood cells (RBC) are swelled to spheres with a hypotonic solution. Dense cells resist becoming spheres and are detected by their low volume and high hemoglobin concentration. However, the number of dense cells might be underestimated because reversibly sickled cells are capable of undergoing the sphering and will not be detected. In addition, the hypotonic solution can lyse the cells. Finally, RBC counters cannot detect cells on the basis of specific cell markers, which can be used to define cell types and cell morphology and activation status (platelets). Conventional flow cytometry can detect cell markers, but yields little information on morphology and cannot detect dense cells. Here, we used the ImagestreamX Flow Cytometer (Amnis) to analyze SCD blood. In addition to providing information available with conventional cytometers, this instrument provides an image of each cell analyzed, thus allowing for detailed morphological assessment of a large population of cells. We analyzed 5 patients. All were outpatients not suffering from acute complications. Blood was collected by venipuncture into citrate anticoagulant, stained with antibodies or other reagents, and then fixed in 4% paraformaldehyde. We evaluated the blood for cell numbers and morphology, reticulocytes, dense cells, platelet-monocyte aggregates, phosphatidylserine exposure, and platelet activation status. The blood from all of the SCD patients displayed characteristics not found in control blood. We could clearly distinguish RBC morphologies corresponding to sickle cells, dense cell and reticulocytes. Reticulocytes, identified by CD71 positivity, often displayed a "puckered" morphology, as previously seen in electron micrographs. We calculated the percentage of RBCs that were sickled based on the shape ratio of > 2 (length along the long axis/maximum thickness along the short axis). The sickle cell percentage was 1.4±0.5% (normal 0%) out of total RBC population. We also evaluated dense cell morphology after separating the cells on a percoll density gradient. The cells appeared flattened and "deflated", clearly indicating their loss of intracellular fluid. We also analyzed platelet activation status based on staining for P-selectin, the activated form of integrin aIIbb3 (PAC-1 antibody), and phosphatidylserine exposure. Platelets staining positively for these markers also demonstrated morphological evidence of activation: shape change and filopodia extension. Platelet-monocyte aggregates were higher in all of the patients than in controls (0.036% vs 0%) and were easily distinguished from coincident events by morphology. The number of platelets associated with individual monocytes varied from 1 to 3. Other heterotypic cell aggregates were rare. In summary, imaging flow cytometry provides a powerful tool for the analysis of blood in SCD. The technique allows cell population analysis like conventional cytometry, while yielding detailed morphological information on many parameters of relevance in the disease. Further, the morphological assessment avoids many of the potential artifacts arising from blood film preparation and allows an unbiased assessment of the results. Disclosures Konkle: Baxalta: Consultancy, Research Funding; Biogen: Consultancy, Research Funding; CSL Behring: Consultancy, Other: IDMC chair; Pfizer: Other: IDMC member; Octapharma: Research Funding; Novo Nordisk: Consultancy.

Fine Temporal Mapping of Plasmodium Falciparum growth Inhibition in Hypoxic Sickle Cell Trait Erythrocytes

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 924-924
Author(s):  
Natasha Archer ◽  
Nicole Petersen ◽  
Martha Clark ◽  
Manoj Duraisingh
Keyword(s):  
Flow Cytometry ◽  
Carbon Monoxide ◽  
Dna Replication ◽  
Oxygen Tension ◽  
Sickle Cell ◽  
Sickle Cell Trait ◽  
High Oxygen ◽  
Low Oxygen ◽  
Trophozoite Stage ◽  
Sickle Hemoglobin

Abstract Background: Despite the global acceptance of Haldane's hypothesis that individuals with sickle cell trait (AS) are protected against malaria, an exact mechanism of resistance remains unknown. Several theories to explain the resistance such as increased splenic destruction, impaired hemoglobin digestion, reduced cytoadherence and specific translocation of host specific microRNAs have been proposed. Here we carefully examine the influence of hypoxia and its relation to parasite cytoadherence and growth in AS and normal (AA) human red blood cells (RBCs). Materials and Methods: To assess the influence of oxygen tension on parasite proliferation in AS RBCs, we followed the proliferation of tightly synchronized P. falciparum within AS and AA RBCs at 1, 5, 10, and 17% oxygen by flow cytometry and microscopy. Carbon monoxide (CO) studies were performed by introducing 100% CO gas into our enclosed chamber system every 21 hours to prevent sickling at low oxygen. Results: Flow cytometry assessment of DNA content confirms the absence of DNA replication within parasites in AS erythrocytes at low oxygen, however normal DNA replication is observed in the presence of high oxygen tension and when CO is introduced at low oxygen. Moving parasites from high to low oxygen tension greatly influences the extent of parasite maturity; parasites moved at 16 and 20 hours post invasion (hpi) do not mature beyond that of the late ring/early trophozoite stage while parasites moved at 24, 28, and 32 hpi develop increasingly more merozoites. Conclusion: Here, we show that in hypoxic conditions parasites in AS erythrocytes stall at the late ring/early trophozoite stage and do not replicate their DNA. We also show that treatment of AS erythrocytes with high oxygen tension or carbon monoxide, a potent inhibitor of sickle hemoglobin polymerization, reverses this phenotype. We propose that the mechanism of AS protection is largely due to P.falciparum's inability to digest polymerized sickle hemoglobin once the parasite sequesters in the hypoxic microvasculature. This is likely a key driver of the reduced parasite densities observed in actual infections in AS individuals. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Alcohol Induced Alterations of Red Cell Morphology in Carriers of the Sickle Cell Trait

1969 ◽  
Vol 16 (1) ◽  
pp. 157-160 ◽  
Author(s):  
Shirley Rubler ◽  
Renee Altman Fleischer ◽  
Eleanor Roth
Keyword(s):  
Sickle Cell ◽  
Cell Morphology ◽  
Sickle Cell Trait ◽  
Red Cell

Flow cytometry using spectrally encoded confocal microscopy

2010 ◽  
Vol 35 (13) ◽  
pp. 2218 ◽  
Author(s):  
Lior Golan ◽  
Dvir Yelin
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Spectrally Encoded

Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1902-1902
Author(s):  
Yamaja Setty ◽  
Suhita Gayen Betal ◽  
Jie Zhang ◽  
Nigel S Key ◽  
Marie Stuart
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Dependent Manner ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Tnf Α ◽  
Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P<0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p<0.000001), and reticulocyte count (r=0.60, p<0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 905-905
Author(s):  
Julia E. Brittain ◽  
David Manly ◽  
Leslie V. Parise ◽  
Nigel Mackman ◽  
Kenneth I. Ataga
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Monocytic Cells ◽  
Stress Erythropoiesis

Abstract Abstract 905 Introduction: Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD. Objective: With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients. Methods: Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA. Results: We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (rspearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (rspearman = 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD. Conclusion: Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD. Disclosures: No relevant conflicts of interest to declare.

Increased Platelet-Derived CD40L May Modulate Endothelial Cell ICAM-1 Expression and Interact With Leukocytes In Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 974-974
Author(s):  
Vanessa Tonin Garrido ◽  
Renata Proença-Ferreira ◽  
Venina M. Dominical ◽  
Marcos André Cavalcanti Bezerra ◽  
Aderson S. Araujo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Repeated Measures ◽  
Conflicts Of Interest ◽  
Endothelial Activation ◽  
Surface Expression ◽  
Platelet Surface

Abstract Background Vaso-occlusive events are a major cause of morbidity in sickle cell anemia (SCA) and attributable to the abnormal adhesion of red cells and leukocytes to the endothelium. Platelets may contribute to the chronic inflammation and endothelial activation that initiates the vaso-occlusive process. We hypothesized that platelet-associated CD40 ligand (CD40L) may contribute to platelet-mediated inflammatory responses in SCA. Aims This study evaluated the platelet (PLT) release of CD40L, the expression of its receptor (CD40) on platelets, neutrophils, lymphocytes and monocytes of control individuals (CON) and SCA patients, and also the ability of platelet-derived CD40L to activate endothelial cells. Methods IL-8, soluble ICAM-1, VCAM-1 and CD40L were determined in PLT-free plasma or the supernatant of stimulated (ADP or Collagen) and unstimulated PLTs (2•10⁸/mL in Kreb’s buffer), from CON individuals and steady-state SCA patients, by ELISA. Flow cytometry was used to analyze CD40 expression on platelets, neutrophils, lymphocytes and monocytes from the peripheral blood of the study’s subjects. Human umbilical vein endothelial cells (HUVECs) were cultured (1x106cells/well; 37°C, 5% CO2) together with PLTs (3x108PLTs/well) from CON individuals or steady-state SCA patients for 24h, 37°C, 5%CO2, in the presence, or not, of blocking antibodies against CD40L. After incubation, PLTs were removed and HUVECs analyzed by flow cytometry for CD54 (ICAM-1) surface expression. Results SCA individuals presented elevated levels of plasma CD40L (724.4± 55.7 pg/ml; n=90) compared to CON (241.5±34.6 pg/ml; n=41; P<0.0001) and these levels correlated with PLT counts (rs=0.255; P=0.015). No correlation was found between plasma CD40L and plasma IL-8, ICAM-1 or VCAM-1. PLT release of CD40L (90 min, 37°C, 5%CO2) was evaluated; PLTs of SCA patients released higher quantities of CD40L (8347±1464 pg/108 PLTs; n=10) than PLTs of CON individuals (3652±568 pg/108 PLTs; n=5; P=0.019). CD40L release from SCA PLTs was augmented by incubation with collagen (P<0.001), but not ADP. Expression of the CD40 receptor on the platelet surface was elevated in the SCA group (52.4±2.7% positive cells; n=23), compared to the CON group (36.8±3.7% positive cells; n=9; P=0.005). The surface expression of CD40 was also elevated on neutrophils (SCA, 10.4±1.5% positive cells, n=14; CON, 5.5±1.1% positive cells, n=13; P=0.03), lymphocytes (SCA, 8.3±0.8% positive cells, n=16; CON, 3.6±0.4% positive cells, n=14; P<0.001) and monocytes (SCA 69.6±5.9% positive cells, n=16; CON, 49.9±5.8% positive cells, n=14; P=0.03) of SCA patients, compared to controls. ICAM-1 expression on the surface of HUVECs (Basal expression 32.8±1.8%, n=11) was significantly increased following incubation with SCA PLTs (54.0±4.8%, n=11, p<0.0001) and slightly augmented after incubation with CON PLTs (40.8±3.1%, n=11, p<0.05; Repeated measures ANOVA). Interestingly, when HUVECs and SCA PLTs were incubated with a blocking antibody against CD40L, the increase in ICAM-1 expression was significantly reversed on HUVECs (HUVECs, 28.1±0.2%, n=6; HUVECs+SCA PLTs, 42.0±3.3%, n=6; HUVECs+SCA PLTs+anti-CD40L 28.9±1.5%, n=6; P<0.01). Conclusions Plasma levels and platelet release of CD40L were found to be significantly elevated in SCA, in association with increased expressions of the CD40 receptor on SCA PLTs, neutrophils, lymphocytes and monocytes, possibly indicating a CD40L-mediated crosstalk between platelets and leukocytes in SCA. Platelets from SCA patients can induce adhesion molecule expression on the surface of endothelial cells in vitro, and this up-regulation may be modulated by platelet-derived CD40L. Results suggest that the CD40/CD40L pathway may be altered in SCA and that platelets may participate in this up-regulation. Given the potent inflammatory effect of this cytokine, a role for platelets and this cytokine in endothelial activation, inflammation and consequent vaso-occlusion, is likely. Disclosures: No relevant conflicts of interest to declare.

Mitophagy Induction Is a Potential Therapeutic Approach for Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 267-267
Author(s):  
Ramasamy Jagadeeswaran ◽  
Benjamin Alejandro Vazquez ◽  
Vinzon Ibanez ◽  
Maria A Ruiz ◽  
Robert E Molokie ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Therapeutic Approach ◽  
Research Funding ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Blood Disorder ◽  
In Vivo Treatment

Abstract Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. A single point mutation of the sixth amino acid of β-globin causes glutamic acid to be replaced by valine, rendering the hemoglobin susceptible to polymerization when deoxygenated. SCD patients suffer from the wide variety of disease manifestations including chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. In addition to the HbS polymerization-mediated rigid and fragile sickle-shaped red blood cell (RBC) formation, an excessive formation of intracellular reactive oxygen species (ROS) occurs in SCD red blood cells, which accelerates their hemolysis. This causes the release of ROS, free extracellular hemoglobin, hemin, and inflammatory cytokines that trigger disease progression. We analyzed levels of ROS in SCD patient RBCs and observed a higher fraction of intracellular ROS positive RBC in SCD (HbSS) compared to control (HbAA) RBC of adults [Control (HbAA): 7.1%± 1.4 %, n=11; SCD (HbSS): 25.3 % ± 4.3%, n=9; p<0.0004]. We also made the novel observation that mature RBCs from SCD patients abnormally contain mitochondria as evidenced by flow cytometry analysis of blood samples of 36 SCD patients and 14 normal human control subjects.[Control (HbAA):0.4 % ± 0.04%, n=14; SCD (HbSS): 7.8%± 0.9%, n=30; p<0.0001]. Further subset analysis from SCD patients with HbSC showed mitochondrial retention in their mature RBCs [HbSC: 2.2%± 0.6%,n=6 p<0.01], however to a lesser degree than patients with HbSS. Transmission electron microscopy confirmed the presence of mitochondria in mature RBC of patients with SCD. ROS analysis between mitochondria positive vs. negative fractions showed that mitochondria-positive (TMRM+) RBC fractions have higher levels of ROS compared to mitochondria-negative (TMRM-) RBC fractions. This data strongly suggests that retained mitochondria significantly contribute to the production of ROS in SCD RBCs. Similar to humans, a higher fraction of RBCs of SCD mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) retain mitochondria compared to control mice RBC [Control (HbAA): 0.29% ± 0.18%; SCD (HbSS): 16.68%± 1.9%, p<0.0001]. While investigating RN-1, a lysine specific demethylase-1 (LSD-1) inhibitor, as a HbF inducing agent, we observed that SCD mice treated with RN-1 showed a reduction in the fraction of RBCs which retain mitochondria. Therefore, we investigated mitophagy-inducing drugs as a possible useful therapeutic approach for SCD by administering mitophagy-inducing agent Sirolimus. SCD mice treated with RN-1 (5mg), or Sirolimus (5mg) had a significant decrease in the fraction of mitochondria containing RBCs (RN1: 4.96± 1.0%, p<0.0005; Sirolimus: 6.4% ± 1.8%, p<0.002). We observed a reduction of ROS in mature RBCs coupled with decreased mitochondrial retention in RBCs after in vivo treatment with RN1 or Sirolimus as measured by co-staining of TMRM, APC-conjugated CD71antibody, and CM-H2DCFDA. We also observed a significant improvement in RBC survival after the in vivo treatment with Sirolimus or RN-1. RBC survival was measured by flow cytometry and calculated biotin positive circulating RBCs after 2 days of in vivo labeling [SCD treated with vehicle control: 40 %± 2.6%; SCD treated with RN1 (2.5mg): 69.9 ± 2.6%, p<0.004; Sirolimus (5mg): 67.5% ± 6.1%, p<0.04]. Based on this data, mitophagy-inducing drugs have the potential to be a novel therapeutic approach for the treatment of SCD patients. Disclosures Jagadeeswaran: Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding. Rivers:Acetylon: Research Funding.

scholarly journals Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.

1996 ◽  
Vol 44 (10) ◽  
pp. 1115-1122 ◽  
Author(s):  
S R Hasslen ◽  
A R Burns ◽  
S I Simon ◽  
C W Smith ◽  
K Starr ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Low Temperature ◽  
Cell Morphology ◽  
Spatial Organization ◽  
Molecular Organization ◽  
Surface Molecule ◽  
Surface Distribution ◽  
Surface Molecules ◽  
Molecular Expression

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.

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