Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles Jan Lötvall Andrew F. Hill Fred H. Hochberg Edit I. Buzás Dolores Di Vizio Chris Gardiner Yong Song Gho Igor V. Kurochkin Suresh Mathivanan Peter J. Quesenberry Susmita Sahoo Hidetoshi Tahara Marca H. M. Wauben Kenneth W. Witwer Clotilde Théry

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Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles 2014 Journal of Extracellular Vesicles 10.3402/jev.v3.26913 lotvall2014-minimal-experimental-requirements lotvall2014-minimal-experimental-requirements.txt, the project's staged copy b5fe2b4a43ab6eafdc83d79a7b1c85e219b77379 cc-by-nc biology

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0 spans located by searching the stored text for the quote's exact words, allowing any whitespace between them extracellular vesicles, and what a study must report before it may attribute a cargo or a function to them rather than to whatever co-isolated with them Quotation withheld because this record is not cleared for redistribution. what minimal evidence licenses the claim that the material in the tube is EVs, when no method produces a pure EV fraction Quotation withheld because this record is not cleared for redistribution. a minimal set of biochemical, biophysical and functional standards, published as an ISEV position statement, together with an explicit admission that the field cannot yet name markers that would distinguish its own subtypes Quotation withheld because this record is not cleared for redistribution. method Quotation withheld because this record is not cleared for redistribution. secreted membrane-enclosed vesicles that co-isolate with non-vesicular carriers of the same cargo - protein complexes and lipoproteins Quotation withheld because this record is not cleared for redistribution. expert-consensus expert-consensus Quotation withheld because this record is not cleared for redistribution. extracellular RNA travels in NON-VESICULAR carriers too - AGO2 protein complexes, HDL and LDL - so a functional result attributed to EVs may belong to something else in the same preparation Quotation withheld because this record is not cleared for redistribution. polymer precipitation, used by commercial kits, does not isolate EVs exclusively and is LIKELY to co-isolate RNA-protein complexes - the named case of a method whose output does not match its label Quotation withheld because this record is not cleared for redistribution. THE FIELD'S MOST-USED WORD HAS NO AGREED MEANING, SAID IN THE FOUNDING GUIDELINE: `The term “exosomes” is the most commonly used word to designate any type of EV, and this has become a “buzz term” for EV-related science. The ACTUAL MEANING of this word, however, is NOT UNIVERSALLY ACCEPTED`. The word is simultaneously the most frequent and the least agreed. Quotation withheld because this record is not cleared for redistribution. AND THE REASON NO MARKER CAN SETTLE IT IS STATED AS IMMATURITY, NOT DIFFICULTY: `the field of EV research has not matured to the point that we can propose a list of EV-specific “markers” that distinguish subsets of EVs from each other`. The categories are in use before anything can tell them apart. Quotation withheld because this record is not cleared for redistribution. THE PROTOCOL CHANGES THE OBJECT, STATED IN 2014: `the composition of recovered EVs vary vastly according to the protocols used`. doyle2019 demonstrates the same thing five years later as `different isolation methods ... from the same cell type results in different proteomic profiles`. Quotation withheld because this record is not cleared for redistribution. THE FIELD'S MOST-USED WORD HAS NO AGREED MEANING, and its own society says so: `exosomes` is `the most commonly used word to designate any type of EV` and `has become a BUZZ TERM`, whose `actual meaning ... is not universally accepted` Quotation withheld because this record is not cleared for redistribution. THE FIELD CANNOT NAME THE MARKERS ITS OWN CATEGORIES WOULD NEED: it `has not matured to the point that we can propose a list of EV-specific markers` distinguishing EVs budded from the membrane from those of endosomal origin Quotation withheld because this record is not cleared for redistribution. the proteins that were taken for markers are `exosome-ENRICHED` and not `exosome-SPECIFIC` - an enrichment describes a tendency and cannot draw a boundary, which is precisely what the names had been doing with it Quotation withheld because this record is not cleared for redistribution. there is no consensus gold-standard isolation method and therefore no optimal one to prescribe - the document declines to name a method rather than naming a weak one Quotation withheld because this record is not cleared for redistribution. one of the field's terms could not even be COUNTED: `microparticle` was excluded from the term-usage figure because it is massively used for non-vesicles Quotation withheld because this record is not cleared for redistribution. THE DOCUMENT CALLS ITSELF AN EDITORIAL: `the minimal requirements presented in this editorial`. It is cited ever after as the first of a numbered guideline series, a status conferred on it later by its successor rather than claimed here. Quotation withheld because this record is not cleared for redistribution. investigators are asked to report proteins NOT expected to be enriched in EVs - Table I group 3 - alongside the ones that are, in at least a semi-quantitative manner and for three or more proteins eliminates: a preparation characterised only by what it was hoped to contain, which cannot reveal co-isolation of other intracellular origins · outcome: absent-declared-unreported Quotation withheld because this record is not cleared for redistribution. the EV-containing fluid must be collected gently, limiting cell disruption eliminates: intracellular vesicles released by mechanical damage being counted as extracellular ones - the document says the term 'EVs' MAY NOT BE APPROPRIATE for material isolated that way · outcome: absent-declared-unreported Quotation withheld because this record is not cleared for redistribution. the field's own term usage is counted from PubMed by year, with non-EV senses of 'exosome' and 'microvesicle' manually removed eliminates: an impression about which words the literature uses, replaced by a count - and the count is honest about what it could NOT measure · outcome: partial Quotation withheld because this record is not cleared for redistribution. whether any isolation method yields a pure EV fraction, or whether one can be named as the standard eliminates: nothing - the document reports that a totally pure fraction is technically challenging to obtain and that there is no consensus gold standard · outcome: failed Quotation withheld because this record is not cleared for redistribution. extracellular vesicles extracellular vesicles Quotation withheld because this record is not cleared for redistribution. exosomes exosomes Quotation withheld because this record is not cleared for redistribution. microvesicles microvesicles Quotation withheld because this record is not cleared for redistribution. markers markers Quotation withheld because this record is not cleared for redistribution. lipoproteins lipoproteins Quotation withheld because this record is not cleared for redistribution. gold-standard gold-standard Quotation withheld because this record is not cleared for redistribution. buzz term buzz term Quotation withheld because this record is not cleared for redistribution. extracellular vesicles extracellular vesicles Quotation withheld because this record is not cleared for redistribution. extracellular vesicles, 2014 - AS DEFINED HERE BY ENUMERATION, not by property: `exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets`. The 2023 statement replaces this with three properties (released, bilayer-delimited, non-replicating); THE LIST CAME FIRST AND THE PROPERTY CAME AFTER Quotation withheld because this record is not cleared for redistribution. exosome, 2014 - A BUZZ TERM BY ITS OWN SOCIETY'S ACCOUNT. Used for any EV in practice, defined by endosomal origin in principle, and the isolated material `generally contains a mixture of EVs`. The 2023 statement discourages the word; this one only records that its meaning is not agreed Quotation withheld because this record is not cleared for redistribution. `exosome-enriched` versus `exosome-specific` - THE DISTINCTION THE WHOLE PROBLEM TURNS ON, drawn here in 2014. A marker that is merely enriched cannot separate subsets; the field's subtype names had been treating enrichment as though it were specificity Quotation withheld because this record is not cleared for redistribution. the 2014 HOPE - `we hope to eventually be able to provide specific markers and characteristics of EV subtypes`. READ AGAINST MISEV2023, WHICH REPORTS NO UNIVERSAL MARKERS AND DISCOURAGES THE TERMS, this is a field holding a definition open for nine years and then closing it the other way Quotation withheld because this record is not cleared for redistribution. `gold-standard` DENIED, WITH THE CONSEQUENCE DRAWN: `Quotation withheld` The absence of agreement is treated as barring a claim, not merely as an open question. Quotation withheld because this record is not cleared for redistribution. A SOCIETY NAMES A CATEGORY AND THE LITERATURE DOES NOT FOLLOW: `the term “extracellular vesicles,” chosen as generic term at creation of ISEV in September 2011, is steadily growing` - while `from 2004 onwards, the term “exosome” has become the most often used`. A dated act of naming, and a measured record of it not taking. Quotation withheld because this record is not cleared for redistribution. critiques → commercial EV/exosome isolation kits and polymer precipitation methods, on the ground that they do not isolate EVs exclusively and co-isolate RNA-protein complexes Quotation withheld because this record is not cleared for redistribution. critiques → the use of 'exosome' as a category label, on the ground that its meaning is not universally accepted and the isolated material is a mixture Quotation withheld because this record is not cleared for redistribution. critiques → attributing a cargo or function to EVs from a preparation that also contains AGO2 complexes and lipoproteins carrying the same extracellular RNA Quotation withheld because this record is not cleared for redistribution. exosomes —is-a→ extracellular vesicles exosomes extracellular vesicles strength of the relation: 1 · note: REPLACES the withdrawn 'part-of' edge on the same pair and the same sentence. The relation is class membership: the evidence names a class and lists its members. Quotation withheld because this record is not cleared for redistribution. microvesicles —is-a→ extracellular vesicles microvesicles extracellular vesicles strength of the relation: 1 · note: REPLACES the withdrawn 'part-of' edge on the same pair and the same sentence. The relation is class membership: the evidence names a class and lists its members. Quotation withheld because this record is not cleared for redistribution. A SOCIETY CALLS ITS FIELD'S MOST-USED WORD A BUZZ TERM. `exosomes` designates any type of EV in practice and its actual meaning is NOT UNIVERSALLY ACCEPTED A SOCIETY CALLS ITS FIELD'S MOST-USED WORD A BUZZ TERM. `exosomes` designates any type of EV in practice and its actual meaning is NOT UNIVERSALLY ACCEPTED Quotation withheld because this record is not cleared for redistribution. THE FIELD CANNOT LIST THE MARKERS ITS CATEGORIES WOULD NEED - it `has not matured to the point` where EV-specific markers distinguishing membrane-budded from endosomal EVs can be proposed THE FIELD CANNOT LIST THE MARKERS ITS CATEGORIES WOULD NEED - it `has not matured to the point` where EV-specific markers distinguishing membrane-budded from endosomal EVs can be proposed Quotation withheld because this record is not cleared for redistribution. proteins commonly found in exosome preparations are exosome-enriched, not exosome-specific, since EV subsets share many markers proteins commonly found in exosome preparations are exosome-enriched, not exosome-specific, since EV subsets share many markers Quotation withheld because this record is not cleared for redistribution. common EV isolation protocols do not fully separate non-vesicular entities from EVs common EV isolation protocols do not fully separate non-vesicular entities from EVs Quotation withheld because this record is not cleared for redistribution. the authors hope eventually to provide specific markers and characteristics of EV subtypes the authors hope eventually to provide specific markers and characteristics of EV subtypes Quotation withheld because this record is not cleared for redistribution. from 2004 the term `exosome` overtook every alternative in the literature, while `extracellular vesicles` - chosen as the generic term at ISEV's founding in 2011 - was only steadily growing from 2004 the term `exosome` overtook every alternative in the literature, while `extracellular vesicles` - chosen as the generic term at ISEV's founding in 2011 - was only steadily growing Quotation withheld because this record is not cleared for redistribution. ONE OF THE FIELD'S OWN TERMS COULD NOT BE COUNTED: `microparticle` was excluded from the usage figure because it is massively used to refer to non-vesicles ONE OF THE FIELD'S OWN TERMS COULD NOT BE COUNTED: `microparticle` was excluded from the usage figure because it is massively used to refer to non-vesicles Quotation withheld because this record is not cleared for redistribution. secreted membrane-enclosed vesicles, collectively named - including exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets. A definition by enumeration extracellular vesicles Quotation withheld because this record is not cleared for redistribution. the most commonly used word for any type of EV, whose actual meaning is not universally accepted - called a buzz term by its own society exosomes Quotation withheld because this record is not cleared for redistribution. or more proteins, reported semi-quantitatively, as the minimum characterisation of any EV preparation 3 Quotation withheld because this record is not cleared for redistribution. the year from which `exosome` became the most-used term in the EV literature, against `extracellular vesicles` chosen as the generic term in 2011 2004 Quotation withheld because this record is not cleared for redistribution. semi-quantitative semi-quantitative Quotation withheld because this record is not cleared for redistribution. markers markers Quotation withheld because this record is not cleared for redistribution.

The full text of this article is not redistributable, so it is not included here. The evidence sentences are withheld with it; the tag layer and its grounding status remain. The article itself is at https://doi.org/10.3402/jev.v3.26913.