Flow cytometric quantitation of red blood cell vesicles in thalassemia

Pattanapanyasat, Kovit; Noulsri, Egarit; Fucharoen, Suthat; Lerdwana, Surada; Lamchiagdhase, Pornvaree; Siritanaratkul, Napadol; Webster, H. K.

doi:10.1002/cyto.b.10064

Cited by 88 publications

(83 citation statements)

References 39 publications

(55 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The vesicles contained Hb but were almost completely devoid of the membrane cytoskeletal polypeptides, spectrin, ankyrin and band 4AE1. We, and others, have reported that circulating vesicle levels are also observed in patients with other haemolytic anaemias (Wagner et al, 1986;Setty et al, 2000;Westerman et al, 2002;Pattanapanyasat et al, 2004). The effects of vesiculation include loss of RBC surface area and microspherocyte formation (Allan et al, 1982), and simulates the vesiculation observed in senescent and apoptotic normal cells .…”

mentioning

confidence: 66%

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

et al. 2008

View full text Add to dashboard Cite

SummaryLevels of circulating red blood cell (RBC)-derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6-fold greater in SCA and 4-fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40-50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)-treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

show abstract

mentioning

confidence: 66%

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

et al. 2008

View full text Add to dashboard Cite

show abstract

“…The MP in plasma were analyzed by flow cytometry using a modification of a previously described method: 22 25 μL of plasma diluted 1:1 with PBS-glucose 5mM were analyzed using anti-CD41 (BD, Franklin Lakes, NJ, USA) and anti-glycophorin-A (Dako, Denmark), both diluted 1:10.…”

Section: Analysis Of Microparticlesmentioning

confidence: 99%

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

Ferru

Pantaleo

Carta³

et al. 2013

Haematologica

View full text Add to dashboard Cite

© F e r r a t a S t o r t i F o u n d a t i o ndifferent genetic and clinical status provided new insights into the pathogenic role of circulating MP and possible interventions to control their amount in thalassemia. MethodsUnless otherwise stated, all materials were obtained from Sigma-Aldrich, St. Louis, MO, USA. Additional information about the methods are provided in the Online Supplementary Data. Treatment of red blood cellsVenous blood was drawn from 12 healthy individuals, eight non-splenectomized TI patients and six splenectomized TI patients. All subjects provided written, informed consent before entering the study. The study was approved by the local Ethics Committee and conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.Clinical analyses were performed by routine laboratory tests at the Policlinico Hospital (Milan, Italy). Blood anti-coagulated with heparin was stored in citrate-phosphate-dextrose with adenine (CPDA-1) prior to its use. RBC were washed three times with phosphate-buffered saline (PBS: 137mM NaCl, 2.7mM KCl, 8.1mM K 2 HPO 4 , 1.5mM KH 2 PO 4 , pH 7.4) containing glucose 5 mM to obtain packed RBC.To stimulate HMC formation, RBC from healthy donors were suspended at a hematocrit of 30% and incubated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 mM) of phenylhydrazine at 37 °C for 4 h. To test the antioxidant effect in MP release we incubated 200 μM of 2-mercaptoethanol in the presence of 1 mM phenylhydrazine. When necessary, RBC were pretreated with Syk kinase inhibitors (10 μM Syk inhibitors II and 10 μM Syk inhibitors IV, Calbiochem, Darmstadt, Germany), for 1 h at 37 °C in the dark. Each reaction was terminated by three washes with PBS-glucose. For all protocols described, untreated controls were processed identically except that the stimulant/inducer was omitted from the incubation. Red blood cell membrane preparationStandard hypotonic membranes were prepared, as previously described, 20 and stored frozen at -80°C until use. Membrane protein content was quantified using the DC Protein assay (Biorad, Hercules, CA, USA). Analysis of microparticlesThe MP in plasma were analyzed by flow cytometry using a modification of a previously described method:22 25 μL of plasma diluted 1:1 with PBS-glucose 5mM were analyzed using anti-CD41 (BD, Franklin Lakes, NJ, USA) and anti-glycophorin-A (Dako, Denmark), both diluted 1:10. Assay of hemichromesHMC were quantified by measuring heme absorbance (Abs) using the following equation:HMC= -133xAbs577 -114xAbs630 + 233xAbs560, and expressed as nMoles/mL of solubilized membranes. 23 Microparticle isolationTo induce MP release in vitro, RBC from each volunteer and phenylhydrazine-treated RBC in PBS (30% hematocrit) were incubated as previously described. 24 Electrophoresis and immunoblottingMembrane and MP proteins were solubilized in Laemmli buffer 25 under reducing [2% (w/v) dithiothreitol] or non-reducing conditions at a volume ratio of 1:1. Sodium dodecylsulfate polyacrylamide gel electropho...

show abstract

“…Informed consent was provided according to the Declaration of Helsinki. Complete blood cell count (Cell-Dyn 370, Abbott Diagnostics) and serum level of LDH were obtained by routine methods (Cobas, Roche); LDH isoenzymes by electrophoresis on agarose gel and specific enzymatic revelations (Interlab G26); plasma-Hb determination by spectrophotometry; erythrocytederived microvesicles by flow cytometry (FACSCalibur, BD Biosciences) 7 ; and haptoglobin by immunoturbidimetry (Olympus AU 640 system). Kruskal-Wallis and Spearman correlation tests were performed using GraphPad Prism 5 (GraphPad Software Inc.).…”

mentioning

confidence: 99%

Lactate dehydrogenase isoenzyme 3 and hemolysis in sickle cell anemia: a possible correlation?

Mecabô

Yamamoto

Biassi

et al. 2015

Blood

View full text Add to dashboard Cite

Flow cytometric quantitation of red blood cell vesicles in thalassemia

Cited by 88 publications

References 39 publications

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

Lactate dehydrogenase isoenzyme 3 and hemolysis in sickle cell anemia: a possible correlation?

Contact Info

Product

Resources

About