Analyses of published methods for the isolation and characterization of salivary extracellular vesicles from the MISEV 2018 perspective. Protocol for a systematic review.
Creators: name: Jadue, Fernanda
name: Collao, Víctor
name: Rojas, Cristián
name: Rivera, José
Publisher: name: Open Science Framework
Year: 2022
Subjects: subject: Medical Sciences
subject: Nanomedicine
subject: Medical Biotechnology
subject: FOS: Medical biotechnology
subject: Medicine and Health Sciences
subject: Oral Biology and Oral Pathology
subject: Nanotechnology
subject: FOS: Nanotechnology
subject: Dentistry
subject: FOS: Clinical medicine
subject: exosomes
subject: extracelullar vesicles
subject: ISEV
subject: microvesicles
subject: MISEV 2018
subject: saliva

description: This is the protocol of the systematic review. The structure of the protocol is based on the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 ADMINISTRATIVE INFORMATION 1. Tittle 1.a. Identification: Analyses of published methods for the isolation and characterization of salivary extracellular vesicles from the MISEV 2018 perspective. Protocol for a systematic review. 1.b. Update: First SR on the subject. 2. Registration: The protocol of this systematic review will be registered on Open Science Framework (www.osf.io9). 3. Authors 3.a. Contact · Felipe Martí (FM) · Universidad de Los Andes, Santiago, Chile · Email address: fmartic@gmail.com · Cristián Rojas (CR) · Universidad de Los Andes, Santiago, Chile · Email address: cristianrojas21@gmail.com · José Tomás Rivera (JTR) · Universidad de Los Andes, Santiago, Chile · Email address: jtrivera@miuandes.cl · Fernanda Jadue (FJ) · Universidad de Los Andes, Santiago, Chile · Email address: fernandajadue@gmail.com · Mailing address: El Peumo 271-20, Lo Barnechea, Santiago, Chile. 3.b. Contributions: FJ is the guarantor. FJ developed the selection criteria and data extraction criteria. All authors contributed to the development of the risk of bias assessment strategy. All authors read, provided feedback and approved the final manuscript. 4. Amendments: In the event of protocol amendments, the date of each amendment will be accompanied by a description of the change and rationale and uploaded to Open Science Framework. 5. Support: This systematic review does not receive funding. INTRODUCTION 6. Rationale There is consensus among the scientific community about a lack of standardization in the development and reporting of extracellular vesicle (EV) research. In general, each group of investigators uses its own method to collect, pre-process and store biofluid samples. And later on, uses its own method to isolate the EVs and characterize them. This has turned into a problem, because generally the methods used are not reported with enough detail, making studies incomparable and impossible to be replicated by other investigators. Besides this, a lack of rigor when characterizing the obtained EVs has been observed. The characterization procedures done by investigators do not cover all of the items that have to be evaluated to assure the obtained EV preparation is free of contaminants (non-EV components), affecting the validity of the conclusions reported by the authors. To address this problem, on 2018 the International Society for Extracellular Vesicles (ISEV) published the Minimal Information for Studies of Extracellular Vesicles (MISEV 2018); a list of minimal requirements studies of extracellular vesicles have to comply with and report to be considered reproducible and that the reported conclusions are valid. Even though this requirements were updated recently (there had previously been a MISEV 2014) and one could think it is not fair to judge previous publications under this new parameters, all points stated are of great relevance, and not complying with them may mean that the study conclusions are not valid. For example, if a publication claims they found an oral cancer biomarker on salivary EVs, but they didn’t assess the purity of the EV preparation they obtained from saliva, it could be that the cancer biomarker they found was actually bound to a compound contaminating the EV preparation (e.g., a lipoprotein) and not really associated with salivary EVs. For this reason, this systematic review aims to analyze publications were they have isolated salivary EVs, from the MISEV 2018 perspective, to know if research in this field complies with the defined standards, and thus if studies are reproducible and the conclusions they have reached are valid. This analysis will establish the strengths of the investigations published until know, so that they can be used by investigators interested in this field. It will also identify weaknesses; which will be a starting point for new research questions that will strengthen this area of knowledge. 7. Objectives Scientific question: ¿What is the degree of compliance that the published methods for the isolation and characterization of salivary extracellular vesicles have with the MISEV 2018 requirements? General objective: To describe the degree of compliance that the published methods for the isolation and characterization of salivary extracellular vesicles have with the MISEV 2018 requirements METHODS 8. Elegibility criteria We will include experimental studies were human salivary EVs have been obtained. Studies in animal saliva, other oral fluids and revisions were no experimental procedures have been done will be excluded. We will consider studies published in English and Spanish until August 20, 2020, with no past time limit. Since one of the purposes of the systematic review is to assess the quality of reporting of the experiments, we will only consider published studies. 9. Information sources Literature search strategies will be developed using medical subject headings (MeSH), Emtree terms and text words related to salivary EVs. We will search MEDLINE (PubMed interface), EMBASE, BIREME and EV-TRACK (knowledgebase created to record experimental parameters of EV-related studies). The literature search will be limited to English and Spanish languages and types of studies that are not reviews. The final search will be done independently by two authors (FJ and FM) on August 20, 2020. Exclusion of studies that don’t fulfill the eligibility criteria will be done by reading the titles, abstracts and/or full texts. 10. Search strategy PubMed search strategy: ((((((("saliva"[MeSH Terms] OR "saliva"[All Fields]) OR "salivas"[All Fields]) OR "saliva s"[All Fields]) OR "salivary"[All Fields]) OR ((("mouth"[MeSH Terms] OR "mouth"[All Fields]) OR "oral"[All Fields]) AND (("fluid"[All Fields] OR "fluid s"[All Fields]) OR "fluids"[All Fields]))) AND (((((((((("exosomal"[All Fields] OR "exosomes"[MeSH Terms]) OR "exosomes"[All Fields]) OR "exosome"[All Fields]) OR "exosomic"[All Fields]) OR (((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "microvesicle"[All Fields]) OR "microvesicles"[All Fields])) OR (((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "microparticle"[All Fields]) OR "microparticles"[All Fields])) OR (((("extracellular vesicles"[MeSH Terms] OR ("extracellular"[All Fields] AND "vesicles"[All Fields])) OR "extracellular vesicles"[All Fields]) OR ("extracellular"[All Fields] AND "vesicle"[All Fields])) OR "extracellular vesicle"[All Fields])) OR ((((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "ectosome"[All Fields]) OR "ectosomes"[All Fields]) OR "ectosomal"[All Fields])) OR "oncosome"[All Fields]) OR (((("extracellular vesicles"[MeSH Terms] OR ("extracellular"[All Fields] AND "vesicles"[All Fields])) OR "extracellular vesicles"[All Fields]) OR ("apoptotic"[All Fields] AND "body"[All Fields])) OR "apoptotic body"[All Fields]))) AND ("english"[Language] OR "spanish"[Language])) NOT "review"[All Fields] EMBASE search strategy: (('saliva' OR 'saliva'/exp OR saliva OR salivary OR 'oral fluid'/exp OR 'oral fluid') AND ('exosome' OR 'exosome'/exp OR exosome OR 'membrane microparticle'/exp OR 'membrane microparticle' OR 'platelet microparticle'/exp OR 'platelet microparticle' OR 'endothelial microparticle'/exp OR 'endothelial microparticle' OR 'microparticle' OR 'microparticle'/exp OR microparticle OR 'microparticles' OR 'microparticles'/exp OR microparticles OR 'ectosome' OR 'ectosome'/exp OR ectosome OR 'oncosome' OR 'oncosome'/exp OR oncosome OR 'apoptotic body'/exp OR 'apoptotic body') AND ([english]/lim OR [spanish]/lim) AND [embase]/lim) AND ('article'/it OR 'article in press'/it OR 'chapter'/it OR 'conference abstract'/it OR 'conference paper'/it OR 'editorial'/it OR 'letter'/it OR 'note'/it OR 'short survey'/it) BIREME search strategy: tw:((tw:(saliva OR "fluido oral")) AND ((tw:(exosoma)) OR (tw:(exosomas)) OR (tw:("vesícula extracelular")) OR (tw:("vesículas extracelulares")) OR (tw:("partícula extracelular")) OR (tw:("partículas extracelulares")) OR (tw:(microvesícula)) OR (tw:(microvesículas)) OR (tw:(micropartícula)) OR (tw:(micropartículas)) OR (tw:("cuerpo apoptótico")) OR (tw:("cuerpos apoptóticos")) OR (tw:(oncosoma)) OR (tw:(oncosomas)) OR (tw:(ectosoma)) OR (tw:(ectosomas)))) AND ( la:("en")) EV-TRACK search strategy: Biofluid: saliva 11. Study records 11.a. Data management: Literature search results will be entered by two authors independently (FJ and FM) in an excel spreadsheet previously designed. One sheet will contain included studies, while another sheet will contain excluded studies with reason for exclusion. Full texts (pdf. files) will be uploaded to a shared file on Zotero reference management software. Because one of the aims of the systematic review is to asses the quality of reporting, experiments that are published more than once (for example in two different journals) will be analyzed individually, because the details reported in each publication could be different. 11.b. Selection process: Two authors (FJ and FM) will independently conduct the search and screen titles and abstracts for the evaluation of the eligibility criteria. If there is any uncertainty, full texts will be read. Any disagreement between the authors will be solved by discussion. If there is no agreement, a third author (JTR) will decide. 11.c. Data collection process: An excel spreadsheet containing all the MISEV 2018 requirements will be created for data extraction. Extraction of the data from the articles will be done in duplicate by two authors independently (FJ y CR). Results of both authors will be compared and any disagreement will be solved by discussion. If there is no agreement, a third author (JTR) will decide. It is possible that some studies work with EVs from other body fluids, tissues or conditioned media, besides saliva. In that case only data concerning saliva will be extracted. We will consider the information published in the articles and supplementary material the authors may cite. In cases were a methodology is not described in detail, but reference is made to it being cited in a previous publication, we will consider the information obtained from the cited publication. If there is missing information for studies with data uploaded on EV-TRACK, the information will be searched there. If there is any difference between the information published in the article and the data entered on EV-TRACK, we will consider the information contained in the article. In case of information not entered on EV-TRACK and articles that are not uploaded on EV-TRACK, we will not contact the authors for missing information, because quality of reporting is one of the items being analyzed. We will only contact authors to clarify information that generated discrepancies between the two authors analyzing the data and that could not be resolved by the third author (JTR). In case we are not able to contact the authors of the article, we will consider the data is missing, because it will be assumed that in the case we wanted to replicate the experiment, not being able to contact the author would have prevented us form doing it. 12. Data items The data that will be extracted corresponds to the items contained in the 6 domains that the MISEV 2018 encompasses: nomenclature, collection and pre-processing, EV separation and concentration, EV characterization, functional studies and reporting. Specific items that will be extracted for each domain are the following and can be classified as mandatory (***), mandatory if applicable (**) and encouraged (*): Nomenclature • Uses the generic term “extracellular vesicle” with demonstration of extracellular (no intact cells) and vesicular (complies with characterization requirements) nature. Or uses the term “extracellular particle” if the extracellular origin can be demonstrated, but the characterization to demonstrate it’s vesicular nature is not satisfied.*** • Complements the name “extracellular vesicle” with some specification about size, density or other parameter of the studied vesciles.* • Specifies the subcellular origin (“microvesicle” from plasma membrane, “exosome” from endosome) demonstrating the subcellular origin.* Collection and pre-processing • Specifies donor status if available (sex, gender, previous consumption of food/water, medication, time of collection, etc.)** • Quantifies the volume of biofluid sample collected by donor.*** • If individual donor biofluid was pooled, specifies the total volume of biofluid used for EV isolation.** • Reports all known collection conditions (time, collection method, if samples were kept on ice, etc.), including additives al time of collection.*** • Specifies pre-treatment to separate major fluid-specific contaminants before EV isolation.*** • Reports temperature and time of biofluid handling before and during pre-treatment.*** • Reports storage and recovery procedures (e.g., thawing) of biofluid before EV isolation (storage temperature, vessel, time; method of thawing or other simple preparation).*** • Reports storage and recovery of EVs after isolation (Temperature, vessel, time, additives, etc.).*** EV separation and concentration • Specifies experimental details of the method used.*** · Centrifugation: reference number of tube(s), rotor(s), adjusted k-factor(s) of each centrifugation step, temperature, freak settings. · Density gradient: nature of matrix, method of generating gradient, reference (and size) of tubes, bottom-up (simple at bottom, high density) or top-bottom (simple on top, low density), centrifugation speed and time (with brake specified), method and volume of fraction recovery. · Chromatography: matrix (nature, pore size), loaded simple volume, fraction volume and number of fractions. · Precipitation: reference of polymer, ratio vol/vol or weight/vol polymer/fluid, time and temperature of incubation, centrifugation time, speed and temperature. · Filtration: reference of filter type (nature of membrane, pore size), time and speed of centrifugation, initial and final volume (in case of concentration). · Antibody-based: reference of antibodies, mass of antibodies per amount of EVs, nature of antibody carrier (bead, surface, etc.), and amount of antibodies per carrier surface. · Other necessary details to allow replication. · Additional steps to concentrate, if any. · Additional steps to wash matrix and/or simple, if any. • Reports category of the chosen EV separation/ concentration method.* · High recovery, low specificity: mixed EVs and non-EV components. · Intermediate recovery, intermediate specificity: mixed EVs with limited non-EV components. · Low recovery, high specificity: subtype(s) of EVs with as little non-EV as possible. · High recovery, high specificity: subtype(s) of EVs with as little non-EV as possible. EV Characterization • Quantification · Reports the volume of biofluid used to isolate EVs.*** · Performs global quantification by at least 2 methods: total protein amount, particle number, lipid amount, expressed per volume of initial fluid.*** · Reports the ratio of the 2 quantification figures.*** • Global characterization · Evaluates the presence of at least one transmembrane or GP-anchored protein localized at plasma membrane or endosomes.*** · Evaluates the presence of at least one cytosolic protein with membrane-binding or association capacity.*** · Evaluates the presence/ absence of at least on expected contaminant.*** · If the study investigates specifically small EVs (less than 200nm), it evaluates the presence of proteins associated with other cellular compartments (different from the plasma membrane and endosomes).** · If the study identifies a functional soluble protein on EVs, in addition to identifying that component, it evaluates it’s possible transmembrane ligands (receptors on EVs).** · Evaluates the topology of the relevant functional components.* • Individual characterization of EVs · Reports images of single EVs by wide-field and close-up (e.g., electron microscopy, scanning probe microscopy, super-resolution fluorescence microscopy, etc.).*** · Uses a non-image-based method that analyses large numbers of single EVs (e.g., NTA, high-resolution flow cytometry, Raman spectroscopy, etc.).*** Functional studies • Performs a dose-response assessment.*** • Performs a negative control: Evaluates function in the biofluid of control donors, as applicable.*** • Performs a quantitative comparison of the functional activity of total biofluid, versus EV-depleted biofluid, versus EVs (after high recovery/low specificity separation) (always considering that contaminants could have been co-isolated).*** • Performs a quantitative comparison of the functional activity of EVs, versus other extracellular particles or fractions (contaminants) (after low recovery/high specificity separation).*** • If EV subtype-specific function is being claimed, they perform the quantitative comparison of the functional activity of the different subtypes of EVs.* • Demonstrates that the functional activity exists in the absence of contact between EV donor and EV recipient cells.* Reporting • Submits methodological details to EV-TRACK (evtrack.org) with EV-TRACK number provided.* • Submits data (proteomic, sequencing, other) to relevant public, curated databases or open-access repositories.*** • Submits data to EV specific databases (e.g., EVpedia, Vesiclepedia, exRNA atlas).* • Tempers EV-specific claims when MISEV requirements cannot be entirely satisfied.** 13. Outcomes y prioritization All outcomes will be assessed by two independent authors (FJ y CR). Any disagreement will be solved by discussion. If there is no agreement, a third author (JTR) will decide. Primary outcome The primary outcome will be the degree of compliance of the studies with the MISEV 2018 requirements. All mandatory (*** and **) items of each domain will be evaluated and classified as “accomplished” or “not accomplished”. When an item is classified as “not accomplished” the author will specify the reason for that classification. Depending on how many items are classified al “accomplished”, each of the 6 domains will be classified as: • Accomplished: All of the items of the domain are accomplished. • Partially accomplished: Some of the items of the domain are accomplished. • Not accomplished: None of the items of the domain are accomplished. To represent the information in a graphic form and thus make visualization of the results easier, a color code will be used: • Accomplished: Green • Partially accomplished: Yellow • Not accomplished: Red Results will be presented for each study and also for the proportion of studies classified in each category (accomplished, partially accomplished, not accomplished) for each of the 6 MISEV 2018 domains. Secondary outcomes Secondary outcomes will be evaluated with the intention of knowing more in detail the characteristics of publications that have studied salivary EVs (types of EVs studied, types of saliva samples that have been used, methods of EV isolation and characterization, etc.). The outcomes that will be evaluated are: • Subtypes of EVs that have been studied: · Exosomes · Microvesicles · Microparticles · Small EVs · Large EVs · Ectosomes · Oncosomes · Apoptotic bodies • Types of saliva that have been studied · Unstimulated whole saliva · Stimulated whole saliva · Unstimulated saliva from a specific salivary gland · Stimulated saliva from a specific salivary gland. • Pre-treatments used to lower the viscosity of saliva prior to EV isolation · Centrifugation · Sonication · Filtration · Dilution · Combination of methods • EV separation and concentration methods that have been used · Differential ultracentrifugation · Density gradient centrifugation · Filtration · Size exclusion chromatography · Precipitation · Immunoaffinity capture · Combination of methods • EV global quantification methods that have been used · Total protein concentration · Particle concentration · Lipid concentration • Protein markers that have been used to characterize salivary EVs. • Contaminants that have been analyzed on salivary EV preparations. • Single EV characterization methods that have been used · Transmission electron microscopy · Scanning electron microscopy · Super-resolution fluorescence microscopy · Nanoparticle tracking analyses (NTA) · High-resolution flow cytometry · Raman spectroscopy 14. Risk of bias in individual studies MISEV 2018 requirements seek that conclusions reported by studies on EVs are supported by the experimental procedures that were done (are valid) and that the reported experimental parameters allow other investigators to replicate the experiments. Because of this, as more MISEV 2018 requirements are met, it is considered that the study has less risk of bias. Thus, it can be argued that the main outcome of this systematic review is assessing the risk of bias of the published studies. For this reason, a separated risk of bias assessment will not be done. The primary outcome results will be extrapolated into a risk of bias classification for each study as follows: • Low risk of bias: All MISEV 2018 domains classified as accomplished (green). • Unclear risk of bias: 1 or more MISEV 2018 domains classified as partially accomplished (yellow), with no domains classified as not accomplished (red). • High risk of bias: 1 or more MISEV 2018 domains not accomplished (red). Results will be presented for each study and globally; presenting the proportion of studies that is classified in each risk of bias category. A graphic representation will be made. 15. Data synthesis 15.a. No quantitative synthesis will be done. 15.b. Doesn’t apply. 15.c. Doesn’t apply. 15.d. A narrative qualitative synthesis will be provided, were results will be presented starting with the primary outcome and continuing with the secondary outcomes in the order they appear in this protocol. The primary outcome will be presented in narrative form and graphically, as explained above. The secondary outcomes will be presented in tables as general results and as a narrative synthesis for a more detailed analysis. We will report results for all included studies irrespective of their risk of bias classification. 16. Meta-bias(es): Doesn’t apply. 17. Confidence in cumulative evidence: Doesn’t apply, because the aim of the systematic review is to evaluate the quality of the studies that have been published on this subject.