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Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics

ABSTRACT: Extracellular vesicle (EV) analysis from blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; 6 preserving and 5 non-preserving) and 3 blood processing intervals (BPI; 1h, 8h and 72h) on defined performance metrics (n=9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a platform to guide future research on EV pre-analytics and further support methodological standardization of EV studies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Pekka Rappu

LAB HEAD: Olivier De Wever

PROVIDER: PXD035199 | Pride | 2023-03-13

REPOSITORIES: Pride

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Project description:Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several kinds of macromolecules implicated in cell communication. EVs have become an important topic in the study of pathogenic fungi, due to their relation with the fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EV cargo, because of plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, little is known about EVs of Botrytis cinerea, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the first isolation and characterization of B. cinerea EVs; and the description of their potential role during the infective process. All the experimental process was conducted in B. cinerea growing in mimimal salt medium supplemented with glucose as a constitutive stage, and deproteinized tomato cell walls (TCW) as virulence inductor. The isolation of EV was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EV fractions were visualised by TEM using negative staining. Proteomic analysis of EV cargo was addressed by LC-MS/MS. The used methodology had allowed, for the first time, the correct isolation of B. cinerea EVs, and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in the morphology under both assayed conditions. GO analysis of EV proteins showed an enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed difference in EV function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, between others. This is the first evidence and characterization of B. cinerea EVs production, showing that EVs are essential in the infection process of B. cinerea and share crucial functions with the conventional secretion pathway.

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Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics

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