TITLE:
Standardization of Sampling for Isolation of Exosome-Like Small-Extracellular Vesicles from Peripheral Blood from Reproductive-Aged Women
AUTHORS:
Jéssica Maria Diehl, Júlia Abbade Tronco, Natália Prearo Moço, Graziela Gorete Romagnoli, Ana Clara Faquineli Cavalcante Mendes de Avila, Juliano Coelho da Silveira, Márcia Guimarães da Silva, Bruna Ribeiro de Andrade Ramos
KEYWORDS:
Exosome-Like Small-Extracellular Vesicles, Plasma, Reproductive-Aged Women
JOURNAL NAME:
Open Journal of Obstetrics and Gynecology,
Vol.8 No.11,
September
27,
2018
ABSTRACT: Exosome-like small-extracellular vesicles (sEVs) are extracellular
vesicles that act in intercellular communication and are involved in several
biologic and pathologic processes. While sEVs increase the stability of their
cargo molecules, there is still a need for standardization of sampling and isolation
of these microvesicles. We aimed to determine the best sampling method for
isolation of sEVs from peripheral blood from reproductive-aged women. Material
and Methods: We included samples of plasma from our biobank collected in
2014 by venipuncture in heparin tubes and stored at -80°C. We also included
blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid
(EDTA) tubes and stored at -80°C for one to two weeks prior processing. All
blood samples were collected from the same nine reproductive-aged female
volunteers. sEVs were isolated from plasma by ultracentrifugation and
filtration and indirectly quantified using Pierce BCA Protein Assay kit.
Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were
performed to confirm the isolation of sEVs. Results and Discussion: TEM
and NTA confirmed the isolation of sEVs. Protein concentration of short-time
stored heparin samples was not statistically different from long-time stored
heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1,
p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2,
p = 0.44). In conclusion, blood samples may be collected using heparin or
EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs
integrity and may be used, especially in comparative studies.