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【Objective】 To investigate the effects of platelet activation pathways on the characterization of platelet-derived extracellular vesicles(PEVs). 【Methods】 Whole blood from healthy donors was prepared into platelet suspension by centrifugation. Platelets were randomly divided into six groups, the ctrl group added no extra stimulus, and the other five groups were treated with collagen, adenosine diphosphate(ADP), thrombin, Ca2+ ionophore and freeze-thaw cycles (F-T) to activate platelets. Platelet-derived exosomes(PEXOs) and microvesicles(PMVs) were isolated by differential centrifugations, and then were determined by nano-flow cytometry and electron microscopy. The protein markers of PEXOs also were identified by western-blot. The protein concentration and content of PEXOs were also detected. 【Results】 CD9, CD81, TSG101 proteins were detected in all of the PEXOs, which had no calnexin. Both PEXOs and PMVs had CD41; PEXOs were cup-holder-like bilayer membrane vesicles under a transmission electron microscope, while PMVs were irregular membranous structure; 85%-95% of PEXOs were<100nm, 87%-94% of PMVs were 100-300nm. The concentration of exosomes and microvesicles in the F-T group was the highest(205.67±65.27 and 102.73±15.48), followed by the Ca2+ ionophore group(44.42±17.07 and 11.4±4.81). Although in the same size range, the numbers of PEVs induced by different activation conditions varied. The protein concentration of PEXOs in the F-T group(1.11±0.51) was higher than that in the control group(0.32±0.39), ADP group(0.41±0.31) and thrombin group(0.38±0.37), while the total protein(125.40±58.32) was higher than that in other three groups(the ctrl group 25.53±25.96 vs ADP group 37.21±15.73 vs thrombin group 36.28±24.18 vs Ca2+ ionophore group 47.09±23.29). 【Conclusion】 The biological characteristics of PEVs are affected by platelets activation pathways, whose ability to induce protein-packing into exosomes may also be relevant to the particle size. The freeze-thaw cycles can induce high concentrations of extracellular vesicles, which may be an ideal method for the preparation of PEVs.
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【Objective】 To optimize the preparation conditions of platelets as biomarkers to ensure the preparation of platelet samples with low activation and good stability. 【Methods】 The platelet count, platelet activation rate and the count of residual leukocytes were compared among the control and four experimental groups with different centrifugation conditions (120g 10 min vs. 120g 15 min vs.120g 20 min vs.240g 20 min)and different formulations of protective solution, i.e.apyrase, dipyridamole, cilostazol and L-arginine (experimental group 1) vs.apyrase, dipyridamole, cilostazol and adenosine (experimental group 2) vs.apyrase, cilostazol, L-Arginine and adenosine (experimental group 3) vs. apyrase, dipyridamole, L-arginine and adenosine (experimental group 4). 【Results】 Platelets obtained by centrifugation at 120 g for 15 min(73.88±5.36) and centrifugation at 120 g for 10 min(77.65±3.07) had higher recovery rates than other experimental groups(52.77±7.86 and 37.71±13.82)(P0.05). The residual leukocytes in the experimental group centrifuged at 120g for 15 min was slightly lower than that in the experimental group centrifuged at 120 g for 10 min, with no statistical differences (P>0.05). The platelet activation rates of four experimental groups were all significantly lower than that of the control within 72 hours (P0.05). 【Conclusion】 For preparing platelets as biomarker samples, the group with solution including apyrase, dipyridamole, cilostazol and L-arginine, and centrifugation at 120 g for 15min, showed the minimum residual leukocytes and the optimum stability within 72 hours.
RESUMO
Activated platelets secrete bioactive molecules and extracellular vesicles. Platelet-derived extracellular vesicles(PEVs), which are rich in proteins, have become important mediators of intercellular communication and cargo exchange, being involved in not only hemostasis and thrombosis, but also in immunity and tissue repair. In past decades, many advances have been made in the isolation, characterization and application of PEVs. Platelet activation modes influence the proteomic features of formed extracellular vesicles. The activation ways may be closely related to functional differences. This review summarized the proteomics and functions of PEVs induced by different platelet activation pathways.
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【Objective】 To establish an indirect ELISA method for detecting IgG, IgM and IgA antibodies against Hepatitis E virus (HEV). 【Methods】 Tn-5 cells were infected with recombinant HEV baculovirus, and HEV-like particles (VLP) were collected and purified for coating antigen. The reaction conditions and methodology of indirect ELISA method were established and evaluated. The prevalence of HEV antibody among blood donors in Xishuangbanna were detected. 【Results】 The collected and purified VLP showed HEV antigenicity. The positive rates of anti-HEV IgG, IgM and IgA in blood donors in Xishuangbanna Prefecture were 18%(90/500), 5.6%(28/500) and 2.6%(13/500), respectively. The recent HEV infection rate was 1.8 % (9/500), and the seroprevalence of hepatitis E was 19.8% (99/500). Of the 13 anti-HEV IgA positive samples, 2 were both anti-HEV IgG and IgM negative. 【Conclusion】 HEV antibody positive is common among blood donors in Xishuangbanna, some of which are recent infections, posing a threat to the safety of blood transfusion. HEV IgA antibody indirect ELISA combined with human HEV IgM antibody detection can improve the detection rate of recent HEV infection.
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【Objective】 To establish an efficient digital PCR method for Babesia detection, so as to provide data reference for the follow-up studies and the evaluation of blood supply safety in China. 【Methods】 18S rRNA conservative gene sequence of Babesia spp. was downloaded from Genbank, and primers were designed according to the common part of the sequence to establish a highly sensitive and absolutely quantitative digital PCR detection method for Babesia detection. A total of 1000 red blood cell samples collected from voluntary blood donors in Mudanjiang City from July 19, 2016 to August 24, 2016 were detected using this new digital PCR method. 【Results】 The established digital PCR method for Babesia detection showed a good repeatability and could steadily detect the mono-copied nuclear acid. The positive rate was 0.2%(2/1 000)by this method. The two blood donors were further confirmed positive by Indirect Immunofluorescence Assay (IFA), and preliminarily judged as asymptomatic infection. 【Discussion】 The digital PCR method for Babesia detection established in this study has high sensitivity and stability, and the its application on blood screening is conductive to reduce the threats of asymptomatic infection to blood safety