Apoptosis detection

Abstract

The present invention provides a method of detecting the presence or absence of apoptotic cells and / or vesicles thereof in a sample, the method comprising: providing a sample; determining a level of extracellular and / or extravesicular surface display of a glycan in the sample; comparing the level of display of the glycan in the sample with a reference value; and detecting the presence of apoptotic cells and / or vesicles thereof, or detecting the absence of apoptotic cells and / or vesicles in the sample when based on the comparison.

Description

[0001] APOPTOSIS DETECTION

[0002] Field of the Invention

[0003] The present invention relates to methods and kits for detecting apoptotic cells and / or vesicles thereof, as well as methods and kits for enriching non-apoptotic and apoptotic cells. The invention also encompasses use of an extracellular surface displayed glycan as an apoptotic cell biomarker.

[0004] Background

[0005] Cell death is a natural part of the recycling of cellular components. Approximately one million nucleated cells in the body die per second, which is equivalent to 1.2kg of cells per day. Most cell death occurs via apoptosis, a programmed cell death pathway, that is designed to minimise extracellular exposure of neighbouring cells to pro-inflammatory signals found within apoptotic cells.

[0006] There are two major intracellular pathways that lead to apoptosis: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by intracellular stress signals such as DNA damage, oxidative stress or nutrient deprivation, which result in the release of mitochondrial cytochrome c and activation of caspase-9. The extrinsic pathway is initiated by binding of extracellular ligands to cell surface death receptors such as Fas, which results in activation of caspase-8. Both pathways converge on caspase-3 activation / cleavage which peaks during the early hours post induction of apoptosis. Cleavage of caspase-3 is an indication of a cell’s full commitment to apoptosis. This intracellular change is represented on the cell-surface as a loss of lipid asymmetry.

[0007] Live cells exhibit a phenomenon known as lipid asymmetry across their plasma membranes. In other words, lipid species are asymmetrically distributed on both surfaces of the plasma membrane. This ensures that both surfaces of plasma membranes have different biophysical properties that influence processes such as signal transduction. During apoptosis, however, lipid asymmetry is lost and phosphatidyl serine, which is mainly present on the intracellular surface of the plasma membrane, is exposed to the extracellular surface of the membrane. Consequently, Annexin V (AnnV) is used as part of a well-known, gold standard method for identifying apoptotic cells because it binds, with high affinity and specificity, to phosphatidyl serine located on the extracellular surface of apoptotic cells. However, a disadvantage of using Annexin V to identify apoptotic cells is that one must wait several hours (after the induction of apoptosis) for it to bind to phosphatidyl serine.

[0008] The present invention seeks to present alternative surface markers of apoptosis which are detectable prior to expression of commonly used biomarkers such as phosphatidylserine that is recognised by Annexin V.

[0009] Statements of the Invention

[0010] According to a first aspect of the invention, there is provided a method of detecting apoptotic cells and / or vesicles thereof in a test sample of cells, the method comprising: providing a test sample; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0011] (i) terminal sialic acid residues,

[0012] (ii) terminal galactose residues,

[0013] (iii) terminal mannose residues,

[0014] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0015] (v) terminal fucose residues,

[0016] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0017] (vii) internal N-acetyllactosamine residues; comparing the level of display of the glycan in the test sample with the level of display in a reference sample; wherein a difference in the level of display of the glycan in the test sample compared to a reference level of display of the glycan in a reference sample is indicative that the test sample comprises one or more apoptotic cells and / or a vesicles thereof.

[0018] The test sample may comprise any cell type. Exemplary cell types include cells of the connective tissue (e.g. a fibroblast), a stem cell (e.g. a mesenchymal stem cell, such as a bone marrow mesenchymal stem cell) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC). Preferably, methods of the invention comprise determining the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0019] (i) terminal sialic acid residues,

[0020] (ii) terminal N-acetylgalactosamine (GalNAc) residues,

[0021] (iii) terminal fucose residues,

[0022] (iv) terminal N-acetylglucosamine (GlcNAc) residues, and

[0023] (v) internal N-acetyllactosamine residues.

[0024] More preferably, methods of the invention comprise determining the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0025] (i) terminal sialic acid residues,

[0026] (ii) terminal N-acetylgalactosamine (GalNAc) residues,

[0027] (iii) terminal fucose residues, and

[0028] (iv) internal N-acetyllactosamine residues.

[0029] According to another aspect, there is provided use of an extracellular and / or extravesicular surface displayed glycan as an apoptotic cell biomarker, wherein the glycan comprises one or more residues selected from the group consisting of:

[0030] (i) terminal sialic acid residues,

[0031] (ii) terminal galactose residues,

[0032] (iii) terminal mannose residues,

[0033] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0034] (v) terminal fucose residues,

[0035] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0036] (vii) internal N-acetyllactosamine residues.

[0037] According to another aspect, there is provided use of a lectin to detect apoptotic cells and / or vesicles thereof or enrich apoptotic cells or non-apoptotic cells and / or vesicles thereof.

[0038] In one embodiment, the invention comprises use of a selection of one or more of MAL- II, SNA, GNA, STL, AAL, UEA-I, S JA, PNA and NPL to detect, enrich, isolate or target apoptotic cells and / or vesicles thereof, or non-apoptotic cells and / or vesicles thereof. A terminal fucose may be, for example, an a(1^2) linked fucose linked (e.g. directly linked) to a galactose, e.g. a galactose that is a more internal relative to said terminal fucose. Some examples include Fuc (al, 2) Gal (pi,3) GlcNAc and / or Fuc (al, 2) Gal (Pl,4), with Fuc (al, 2) Gal (P 1,3) GlcNAc being a preferred example. Said terminal fucose may preferably be referred to as a terminal glycan residue capable of being bound by an agglutinin, more preferably Ulex Europaeus Agglutinin I (UEA-I). Thus, a probe for a terminal fucose, such as an a( 1— >2) linked fucose linked (e.g. directly linked) to galactose (e.g. Fuc (al, 2) Gal (Pl,3) GlcNAc and / or Fuc (al, 2) Gal (pi,4)) may be UEA-1. UEA-1 may be said to be specific for terminal a( 1— >2) fucose linkages linked (e.g. directly linked) to galactose. The term Fuc (al, 2) Gal (P 1,3) GlcNAc may also be presented as Fuc al-2 Gal bl-3 GlcNAc; the term Fuc (al, 2) Gal (Pl,4) may also be presented as Fuc al-2 Gal bl -4 GlcNAc; the term a(1^2) linked fucose may also be presented as Fuc al-2.

[0039] As reflected by the preceding sentence, the skilled person would appreciate that the manner in which certain glycans are presented in writing can vary; and would understand that language such as “Fuc al-2” can be used to “a(l— >2) linked fucose” for convenience, and so on. Such alternative presentations may be used herein.

[0040] A terminal fucose may be an a( 1^6), an a( 1— >2), an a( 1^3), and / or an a( 1— >4) linked fucose (each of said alternatives being linked to an N-acetylglucosamine (GlcNAc), e.g. a GlcNAc that is more internal relative to said terminal fucose). More preferably, a terminal fucose may be an a(1^6) fucose, linked to N-acetylglucosamine (GlcNAc)). Some examples include Fuc (al, 6) GlcNAc and Fuc al-2 Gal [6S] bl-4 Glc; with Fuc (al, 6) GlcNAc being a particularly preferred example. Said terminal fucose may preferably be referred to as a terminal glycan residue capable of being bound by Aleuria Aurantia Lectin (AAL). Thus, a probe for a terminal fucose, such as an a(1^6), an a(1^2), an a(1^3), and / or an a(1^4) fucose (linked to N-acetylglucosamine (GlcNAc), e.g. a GlcNAc that is more internal relative to said terminal fucose), may be AAL. AAL can bind terminal fucose, preferably a( 1— >6) linkages. AAL may be said to recognise fucose (e.g. terminal fucose) stemming from the base of glycans where GlcNAc is more internal relative to said terminal fucose. The terms a(1^6), an a(1^2), an a( 1^3), and / or an a(1^4) linked fucose may also be presented as Fuc al- 6, Fuc al-2 and Fuc al-3 (respectively); the term Fuc (al, 6) GlcNAc may also be presented as Fuc al-6 GlcNAc.

[0041] A terminal sialic acid may be an alpha-2, 3-linked sialic acid, preferably a terminal NeuAc a2-3 Gal b 1-3 GalNAc residue. Said terminal sialic acid may preferably be referred to as a terminal glycan residue capable of being bound by Maackia Amurensis Lectin II (MAL-II). Thus, a probe for a terminal sialic acid (such as an alpha-2, 3-linked sialic acid, for example a terminal NeuAc a2-3 Gal bl -3 GalNAc residue) may be MAL- II.

[0042] A terminal sialic acid may be an alpha-2, 6-linked sialic acid, preferably a terminal NeuAc a2-6 Gal bl-4 GlcNAc residue. Said terminal sialic acid may preferably be referred to as a terminal glycan residue capable of being bound by Sambucus Nigra Lectin (SNA). Thus, a probe for a terminal sialic acid (such as an alpha-2, 6-linked sialic acid, for example a terminal NeuAc a2-6 Gal bl-4 GlcNAc residue) may be SNA.

[0043] A terminal N-acetylglucosamine (e.g. P(l,4)-linked GlcNAc) may be a monomer of N- acetylglucosamine and / or an oligomer of N-acetylglucosamine, such as Chitin and / or (G1CNAC)3. Said terminal N-acetylglucosamine may preferably be referred to as a terminal glycan residue capable of being bound by Solanum Tuberosum Lectin (STL). Thus, a probe for a terminal N-acetylglucosamine (such as a P(l,4)-linked GlcNAc) may be STL.

[0044] An internal N-acetyllactosamine (e.g. LacNAc) described herein may be referred to as a disaccharide having galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P- 1,4 bonds, and examples include poly-LacNAc. Said internal N-acetyllactosamine may preferably be referred to as a terminal glycan residue capable of being bound by Solanum Tuberosum Lectin (STL). Thus, a probe for internal N-acetyllactosamine (such as LacNAc) may be STL. Throughout this specification, where STL is referred to, it is preferred that such references are construed as being in the context of being a glycan that binds (e.g. is capable of binding) internal N-acetyllactosamine, unless the context requires otherwise.

[0045] A terminal mannose may be a terminal alpha- 1, 6-linked mannose, preferably alpha-1, 6- linked Man3GlcNAc2 and / or alpha- 1, 6-linked Man5GlcNAc2. Said terminal alpha-1,6- linked mannose may preferably be referred to as a terminal glycan residue capable of being bound by Narcissus Pseudonarcissus Lectin (NPL). Thus, a probe for a terminal mannose (such as a terminal alpha- 1,6-linked mannose, preferably alpha- 1,6-linked Man3GlcNAc2 and / or alpha- 1,6-linked Man5GlcNAc2) may be NPL.

[0046] A terminal mannose residue may be a terminal alpha-1, 3-linked mannose, preferably alpha-1, 3-linked Man3GlcNAc2. Said terminal alpha-1, 3-linked mannose may preferably be referred to as a terminal glycan residue capable of being bound by Galanthus Nivalis Lectin (GNA). Thus, a probe for a terminal mannose residue (such as a terminal alpha-1, 3-linked mannose, preferably alpha-1, 3-linked Man3GlcNAc2) may be GNA.

[0047] A terminal galactose may be a galactosyl (P-1,3) N-acetylgalactosamine, for example, Fuc al-2( Gal b 1-3 GalNAc al-3) Gal bl-4 GlcNAc. Said terminal galactose may preferably be referred to as a terminal glycan residue capable of being bound by Peanut Agglutinin (PNA). Thus, a probe for a terminal galactose (such as a galactosyl (P-1,3) N-acetylgalactosamine, for example, Fuc al-2( Gal bl -3 GalNAc al-3) Gal bl- 4 GlcNAc) may be PNA.

[0048] A terminal galactose may be Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc, that may preferably be referred to as a terminal glycan residue capable of being bound by Styphnolobium japonicum (Japanese pagoda) lectin (SJA). Thus, a probe for a terminal galactose (such as Fuc al-3 (Fuc al-2 (Gal al-3) Gal bl-4) GlcNAc) may be SJA.

[0049] A terminal N-acetylgalactosamine may be GalNAc bl-4 GlcNAc bl -3 GalNAc bl-4 GlcNAc. Said terminal N-acetylgalactosamine may preferably be referred to as a terminal glycan residue capable of being bound by Styphnolobium japonicum (Japanese pagoda) lectin (SJA). Thus, a probe for a terminal N-acetylgalactosamine (such as GalNAc bl-4 GlcNAc bl -3 GalNAc bl-4 GlcNAc) may be SJA.

[0050] Said lectin “SJA” is referred to at various points (e.g. in the context of various embodiments) throughout this disclosure. For each reference to “SJA” throughout this specification, it is preferred that the SJA is “B-SJA-I”, noting that an alternative subunit of SJA (i.e. B-SJA-II) has also been previously described. The experiments in the examples section made use of B-SJA-I. Advantageously, it may be “bispecific” as indicated by the two paragraphs directly preceding this paragraph, thus can be used for probing two sugars within the “terminal galactose” family.

[0051] The skilled person will understand that the following glycans (mentioned above) can be said to fall within a ‘genus’ of glycans that can be called “terminal galactose”, with the following sugars representing ‘species’ of said genus:

[0052] GalNAc b 1 -4 GlcNAc b 1 -3 GalNAc b 1 -4 GlcNAc; this is referred to herein as a “terminal GlcNac”, e.g. with a GlcNac being understood to represent an amino acid sugar derivative of galactose (hence falling within the genus of “terminal galactose”), this can be bound by SJA;

[0053] Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc; this is referred to herein as a “terminal galactose”, this can (also) be bound by SJA; and

[0054] Fuc al-2( Gal bl -3 GalNAc al-3) Gal bl-4 GlcNAc; this is referred to herein as a “terminal galactose”, this can be bound by PNA.

[0055] Thus, items (ii) “terminal galactose residues” and (iv) “terminal N-acetylgalactosamine (GalNAc) residues” in the above-mentioned ‘first aspect’ (and any corresponding list of any other aspect / embodiment described herein) may optionally be grouped together and referred to as a “a genus of terminal galactose residues”, comprising the ‘species’ listed above. That being said, there are embodiments in which discussing the individual ‘species’ may be advantageous, which will be discussed in more detail below.

[0056] As mentioned above, it is preferred that STL is used for binding internal N- acetyllactosamine (e.g. LacNAc). As such, an embodiment of list (i)-(vi) may optionally be absent “terminal N-acetylglucosamine (GlcNAc) residues”. Thus, the glycan list of the above-mentioned ‘first aspect’ (and any corresponding list of any other aspect / embodiment described herein) may optionally be absent item (vi) “terminal N- acetylglucosamine (GlcNAc) residues”. Indeed any reference to “terminal N- acetylglucosamine (GlcNAc) residues” throughout this disclosure is totally optionally and can optionally be omitted from the associated aspect / embodiment. Thus methods of the invention may comprise determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residue selected from the group consisting of:

[0057] (i) terminal sialic acid residues,

[0058] (ii) terminal galactose residues, (iii) terminal mannose residues,

[0059] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0060] (v) terminal fucose residues, and

[0061] (vi) internal N-acetyllactosamine residues.

[0062] Although the term “terminal N-acetylglucosamine (GlcNAc) residues” may be absent as per the list directly above, the list of course embraces “terminal galactose residues” and “internal N-acetyllactosamine residues” that may happen to comprise a GlcNAc component.

[0063] The level of display one or more than one glycan type may be determined in the method of the invention. For example, as will be discussed in more detail below, the level of one or more glycan may be determined by challenging the test sample with a probe or binding partner having affinity for said one or more glycan (e.g. an appropriate lectin, or appropriate lectins) and detecting a level of biomarker-binding partner complex.

[0064] The invention is based on the observation that apoptotic cells and / or vesicles thereof, and non-apoptotic cells and / or vesicles thereof, differentially display glycans on their cell surface (specifically on the extracellular surface of the membrane). Consequently, known bio-physical separation techniques can be used as part of the invention to detect, enrich, isolate, remove and target apoptotic cells and / or vesicles thereof. The present invention does not require cells to be lysed in order to detect the relevant biomarkers. Thus, in a preferable embodiment, the sample (e.g. test sample) has not been subjected to a cell lysis step (such as mechanical disruption, liquid homogenization, high frequency sound waves (sonication), freeze / thaw cycles, or manual grinding). In a more preferable embodiment, the test sample has not been subjected to a cell lysis step, or to a permeabilization step (e.g. detergent treatment), or to an acid treatment step (most preferably the test sample has not been subjected to any of such cell lysis / permeabilization / acid treatment steps). In a suitable embodiment, a method of the invention is practiced using a sample comprising, or consisting essentially of, intact cells.

[0065] The inventors surprisingly identifying that as soon as about 1 hour, or even earlier, after cells initiate apoptosis, they modify their display of glycans on their extracellular surface, particularly glycans that comprise specific terminal monosaccharide residues and / or internal N-acetyllactosamine residues.

[0066] As discussed in the examples section, such difference has been detected across multiple, and diverse, cell types when undergoing apoptosis. Thus, particularly where the “reference level” is indicative of value for said glycan in a reference sample of non- apoptotic cells (e.g. viable cells), a difference whether ‘higher’ or Tower’ is reliably detectable across multiple cell types. Thus, even at such broad level, simply detecting a “difference” provides a readout that indicates the presence of apoptotic cells. Naturally, the invention may also utilise alternative references, for example the “reference level” may be indicative of value for said glycan in a reference sample of apoptotic cells (preferably apoptosis stage-matched cells). When employing such reference value, detecting ‘no’ difference may indicate the presence of apoptotic cells in the test sample. As an alternative to the ‘wherein’ clause of the above-described first aspect may be: wherein ‘no’ difference in the level of display of the glycan in the test sample compared to a reference level of display of the glycan in a reference sample is indicative that the test sample comprises one or more apoptotic cells and / or a vesicles thereof.

[0067] Taken together, an aspect of the invention may read as: a method of detecting the presence or absence of apoptotic cells and / or vesicles thereof in a test sample (e.g. test sample of cells), the method comprising: providing a test sample; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0068] (i) terminal sialic acid residues,

[0069] (ii) terminal galactose residues,

[0070] (iii) terminal mannose residues,

[0071] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0072] (v) terminal fucose residues,

[0073] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0074] (vii) internal N-acetyllactosamine residues,

[0075] (preferably from the group consisting of (i), (iv), (v), and vii)); comparing the level of display of the glycan in the test sample with the level of display in a reference sample; and determining that the sample comprises apoptotic cells and / or vesicles, or determining that the sample does not comprise apoptotic cells and / or vesicles, when based on the comparison.

[0076] Further details on how individual glycans may be compared with a particular reference value will be provided below.

[0077] An aspect of the invention provides a method of assaying a test sample having or suspected of having apoptotic cells and / or vesicles thereof, the method comprising: providing a test sample; and assaying the test sample for a level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0078] (i) terminal sialic acid residues,

[0079] (ii) terminal galactose residues,

[0080] (iii) terminal mannose residues,

[0081] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0082] (v) terminal fucose residues,

[0083] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0084] (vii) internal N-acetyllactosamine residues;

[0085] (preferably from the group consisting of (i), (iv), (v), and vii)); optionally wherein a test sample having apoptotic cells (and / or vesicles thereof) demonstrates a different level of extracellular and / or extravesicular surface display of a glycan than a sample that lacks apoptotic cells (and / or vesicles thereof), or wherein a test sample that does ‘not’ have apoptotic cells (and / or vesicles thereof) does ‘not’ demonstrate a different level of extracellular and / or extravesicular surface display of a glycan than a sample that lacks apoptotic cells (and / or vesicles thereof).

[0086] The invention may comprise detecting the level of extracellular and / or extravesicular display of a glycan at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, or at least about 3 hours or more after the initiation of apoptosis. The invention may therefore comprise detecting the level of extracellular and / or extravesicular display of a glycan about 10 minutes to about 7 hours or more, about 30 minutes to about 7 hours or more, about 1 hour to about 7 hours or more, about 1 hour to about 6 hours or more, about 1 hour to about 5 hours or more, about 1 hour to about 4 hours or more, about 1 hour to about 3.5 hours or more, about 1 hour to about 3 hours or more, about 10 minutes to about 3 hours or more, or about 1 hour to about 2 hours or more, after the initiation of apoptosis. The method may comprise detecting the level of extracellular and / or extravesicular display of one or more glycans in the test sample about 10 minutes, about 30 minutes, or about 1 hour, after the initiation of apoptosis.

[0087] In other words, methods of the invention may comprise detecting apoptotic cells (and / vesicles thereof) that are at a stage of at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, or at least about 3 hours or more post-initiation of apoptosis. Indeed, methods of the invention may comprise detecting cells (and / vesicles thereof) that are at a stage of up to about 10 minutes, up to about 30 minutes, up to about 1 hour, up to about 2 hours, or up to about 3 hours or more postinitiation of apoptosis.

[0088] The invention may therefore comprise detecting cells (and / or vesicles thereof) that are at a stage of about 10 minutes to about 7 hours or more, about 30 minutes to about 7 hours or more, about 1 hour to about 7 hours or more, about 1 hour to about 6 hours or more, about 1 hour to about 5 hours or more, about 1 hour to about 4 hours or more, about 1 hour to about 3.5 hours or more, about 1 hour to about 3 hours or more, about 10 minutes to about 3 hours or more, or about 1 hour to about 2 hours or more, postinitiation of apoptosis. The method may comprise detecting cells (and / or vesicles thereof) that are at a stage of about 10 minutes, about 30 minutes, or about 1 hour postinitiation of apoptosis.

[0089] Difference of individual glycans (or combinations thereof) representative of such timepoints (e.g. glycans associated with 1-3 hours post-initiation) will be discussed in more detail below.

[0090] At a broad level, the apoptosis follows the phases of: induction (pre-commitment), commitment, and execution (post-commitment) phases during which the cells are eventually degraded. Certain timepoints ‘post-initiation’, described above (e.g. up to and including 6 hours) fall within the “pre-commitment” phase of apoptosis. Thus, reference to “apoptotic cells and / or vesicles thereof’ throughout this disclosure embraces apoptotic cells (and / or vesicles thereof) that are in the “pre-commitment” (e.g. induction) phase of apoptosis. Indeed, it may be preferred to refer to “apoptotic cells and / or vesicles thereof’ throughout this disclosure as apoptotic cells (and / or vesicles thereof) that are in the “pre-commitment” (e.g. induction) phase of apoptosis. Additionally or alternatively, it may be preferred to refer to “apoptotic cells and / or vesicles thereof’ throughout this disclosure as apoptotic cells (and / or vesicles thereof) that are phosphatidylserine negative (PS-); e.g. meaning the cells do not (e.g. yet) display phosphatidylserine on their cell surface / outer plasma membrane. It is known to the skilled person that the exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Cells that are phosphatidylserine positive, PS+ (e.g. meaning the cells ‘do’ display phosphatidylserine on their cell surface / outer plasma membrane) may be said to be cells that are in the commitment and / or execution (post-commitment) phases of apoptosis.

[0091] The ability to detect apoptosis at such an early stage is particularly advantageous and solves a yet further problem in the art of how to detect early (e.g. pre-commitment phase) apoptosis. As mentioned above, a disadvantage of using the gold-standard Annexin V to identify apoptotic cells is that one must wait several hours (after the induction of apoptosis) for it to bind to phosphatidyl serine. Thus, while existing methodology fails to find utility in detect early / pre-commitment apoptosis, such detection is readily achievable by methods of the present invention.

[0092] The invention (e.g., a method according to the invention or disclosure) may be performed by detecting extracellular and / or extravesicular surface display of a glycan comprising terminal sialic acid residues and terminal mannose residues.

[0093] The invention may comprise detecting apoptotic cells (and / or vesicles thereof) that are at about 3%-100% (e.g. about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60- 100%, 70-100%, 80-100% or 90-100%) of the way through the pre-commitment phase of apoptosis. The invention may comprise detecting apoptotic cells (and / or vesicles thereof) that are at about 5-90%, 10-80%, 15-75%, 20-70%, 25-65%, 30-60%, 35-55%, or 40-50% of the way through the pre-commitment phase of apoptosis. The method may be performed by detecting extracellular and / or extravesicular surface display of a glycan comprising one or more terminal monosaccharide residues selected from the group consisting of: terminal sialic acid residues, terminal galactose residues, terminal mannose residues, terminal N-acetylgalactosamine residues, terminal fucose residues and terminal N-acetylglucosamine residues; and / or one or more internal N- acetyllactosamine residues.

[0094] The glycan referred to herein may be a homopolymer or a heteropolymer of monosaccharide residues. While the invention involves assaying the level of “extracellular and / or extravesicular surface display” of a glycan, the skilled person would understand that there are scenarios in which the glycan may first be 'liberated’ from the exterior surface of a cell (and / or vesicle thereof) before its level is determined. Such scenarios still allow for assaying a glycan that has been (e.g. pre-liberation solely for measurement / assay purposes) bound to the exterior surface of a cell or vesicle surface, thus continue to allow for assaying the level of “extracellular and / or extravesicular surface display” of a glycan. That being said, the inventors have succeeded in assaying the glycans as they are bound to the cell (or vesicle thereof) surface, e.g. via an advantageous cell sorting / flow cytometry approach. Thus, it is preferred that the glycan referred to herein is extracellular or extravesicular and thus preferably bound to the exterior surface of a cell or vesicle surface.

[0095] It may be said that the glycan is present as an integral part of the outer membrane of the cell (or outer membrane of a vesicle thereof) in the test sample. The glycan may be bound directly or indirectly. The glycan may, for example, be bound indirectly via a cell surface protein or lipid. Thus, the glycan may be part of a glycoprotein or a glycolipid. The glycan may be branched or linear. In embodiments in which the glycan is branched, either / any branch of the glycan may consist of one or more residues of: (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine residues, and / or (vii) internal N-acetyllactosamine residues.

[0096] In one embodiment, the method comprises detecting, in a test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of: (i) terminal sialic acid residues,

[0097] (ii) terminal galactose residues,

[0098] (iii) terminal mannose residues,

[0099] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0100] (v) terminal fucose residues,

[0101] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0102] (vii) internal N-acetyllactosamine residues; wherein detection of a glycan comprising residues according to one or more of

[0103] (i) to (vii) is indicative that the test sample comprises one or more apoptotic cells and / or a vesicles thereof. Therefore, detection of a glycan comprising residues according to one or more of (i) to (vii) may be indicative that the test sample comprises one or more apoptotic cells and / or a vesicles thereof.

[0104] In another embodiment, the method may comprise; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0105] (i) terminal sialic acid residues,

[0106] (ii) terminal galactose residues,

[0107] (iii) terminal mannose residues,

[0108] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0109] (v) terminal fucose residues,

[0110] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0111] (vii) internal N-acetyllactosamine residues; comparing this level of display determined in the test sample with the level of display in a reference sample that does not comprise an apoptotic cell or vesicle thereof (e.g. does not comprise apoptotic cells or vesicles thereof), wherein a difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample comprises one or more apoptotic cells and / or a vesicles thereof, or wherein ‘no’ difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample does ‘not’ comprise one or more apoptotic cells and / or a vesicles thereof.

[0112] In another embodiment, the method may comprise; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0113] (i) terminal sialic acid residues,

[0114] (ii) terminal galactose residues,

[0115] (iii) terminal mannose residues,

[0116] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0117] (v) terminal fucose residues,

[0118] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0119] (vii) internal N-acetyllactosamine residues; comparing this level of display determined in the test sample with the level of display in a reference sample that does comprise apoptotic cells (and / or vesicles thereof), wherein no difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample comprises one or more apoptotic cells and / or vesicles thereof, or wherein a difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample does not comprise one or more apoptotic cells and / or vesicles thereof.

[0120] Therefore, the method according to the invention may comprise comparing the level of display of the glycan detected in the test sample with a reference level for the glycan in a reference sample that does not comprise apoptotic cells and / or vesicles thereof (or alternatively a reference sample that ‘does’ comprise apoptotic cells and / or vesicles thereof), wherein a difference in the level of display of the glycan in the test sample relative to the reference level of display of the glycan in the reference sample is indicative that the test sample comprises apoptotic cells and / or vesicles thereof.

[0121] Throughout this disclosure, a reference sample that ‘does’ comprise apoptotic cells (and / or vesicles thereof) preferably means that the reference sample is indicative of the level of display of the glycan on apoptotic cells at the same stage (post-initiation) as those cell (and / or vesicles thereof) for which the presence (or absence) is to be detected. For example, where the ‘presence’ of apoptotic cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation are detectable when the level of extracellular and / or extravesicular surface display of a given glycan is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof), then said reference sample is preferably indicative of the level of display of the glycan on apoptotic cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation.

[0122] The skilled person would understand that a “reference sample” is used as a control, and that fundamental characteristics such as the cell type (and species from which it derives) may be matched across the test sample and reference sample e.g. where the test sample is a sample of ‘fibroblasts’ the reference sample is also a sample of ‘fibroblasts’; and where the test sample is of “human” origin, so too is the reference sample.

[0123] The difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be a higher level of display or a lower level of display. A higher level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be indicative that the test sample comprises one or more apoptotic cells and / or vesicles thereof. The difference may be a higher level of display of a glycan comprising residues selected from the group consisting of (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine residues, and (vii) internal N-acetyllactosamine residues. The lower level of display may be a lower level of display of a glycan comprising terminal fucose residues, such Fuc al -2 Gal bl- 4 GlcNAc or Fuc al -2 Gal [6S] bl -4 Glc. The lower level of display may be a lower level of display of a glycan comprising terminal fucose residues, such as an a(1^2) linked fucose linked to a galactose. The lower level of display may be a lower level of display of a glycan comprising terminal fucose residues, such as a Fuc a( 1— >6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc).

[0124] A number of embodiments described directly below (and elsewhere) in this disclosure refer to certain hours (or minutes) e.g. post-initiation of apoptosis, with the referenced hours generally falling within up to 6-7 hours e.g. post-initiation. Such time frames are typical of the usual time from of the “pre-commitment” (e.g. induction) phase of apoptosis e.g. prior to the “commitment” phase. Such timeframes are therefore particularly advantageous for assaying “pre-commitment” phase apoptotic cells. For example, noting an upper limit of the time period (post-initiation) that cells may be in the pre-commitment phase may be 6-7 hours, it would be understood that, where a cell is at a time point of 3 hours post-initiation, the pre-commitment phase is about 50% complete. Or in other words, that the cells are at 50% of the way through the precommitment (induction) phase of apoptosis.

[0125] That being said (and particularly knowing the biological processes are subject the variation), the skilled person would understand that such timeframes are subject to variation depending on variables such as cell type, or culture conditions. For example the timeframe (instead of being e.g. 6-7 hours) of the pre-commitment phase may be longer (or short) depending on the conditions (e.g. experimental conditions) employed when conducting methods of the invention. It is well within the ability of the skilled person to confirm a typical timeframe (of the pre-commitment phase) associated with any given conditions employed when conducting a method of the invention. For example, a sample of cells can be initiated to enter the pre-commitment phase (e.g. via contact with an initiation agent such as staurosporine) and the time from post-initiation and up to the emergence of PS+ cells (indicative of cells progressing to the “commitment" phase) would represent a typical timeframe (of the pre-commitment phase) associated with the particular conditions employed when conducting such assay.

[0126] Thus, after discussing detection of cells that are at certain times (e.g. in minutes or hours) post-initiation below, it is discussed how one could (e.g. alternatively) present the invention in terms of detecting cells are at particular percentages of the way through the pre-commitment (induction) phase of apoptosis.

[0127] In one embodiment, the ‘presence’ of apoptotic cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). It is preferred that said apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells that at up to (and including) seven hours post-initiation (e.g. that are between about !4 an hour to seven hours post-initiation, between about i an hour to seven hours postinitiation, or between about one hour to seven hours post-initiation; as an alternative (or in addition) to defining apoptotic cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0128] Throughout this specification, it is preferred that a “terminal mannose” is Man3GlcNAc2 and / or Man5GlcNAc2 (e.g. detectable via NPL / GNA lectins).

[0129] However, where a decrease (Tower’ level) of said glycan when compared to a reference sample that does ‘not’ comprise an apoptotic cell (or vesicle thereof) has been established to correlate with the ‘presence’ of an apoptotic cell (and / or vesicle thereof) the skilled person will understand that no change (or indeed even a ‘higher level’), when compared to a reference sample that does 'not’ comprise an apoptotic cell (or vesicle thereof) may be indicative of the ‘absence’ of an apoptotic cell (and / or vesicle thereof). Likewise, the skilled person would understand that ‘no’ change (or indeed even a Tower’ level) of said glycan when compared to a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof) may be indicative of the ‘presence’ of an apoptotic cell (and / or vesicle thereof). The skilled person would also understand that an increase (‘higher level’) of said glycan when compared to a reference sample that ‘does’ comprise an apoptotic cell (and / or vesicle thereof) may be indicative of the ‘absence’ of an apoptotic cell (and / or vesicle thereof).

[0130] Thus, in one embodiment, the ‘absence’ of apoptotic cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). It is preferred that said apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells that at up to (and including) seven hours post-initiation (e.g. that are between about !4 an hour to seven hours post-initiation, between about i an hour to seven hours post-initiation, or between about one hour to seven hours post-initiation; as an alternative (or in addition) to defining apoptotic cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0131] In one embodiment, the ‘presence’ of apoptotic cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). It is preferred that said apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells that at up to (and including) seven hours post-initiation (e.g. that are between about ! an hour to seven hours post-initiation, between about ! an hour to seven hours postinitiation, or between about one hour to seven hours post-initiation; as an alternative (or in addition) to defining apoptotic cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0132] In one embodiment, the ‘absence’ of apoptotic cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). It is preferred that said apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells that at up to (and including) seven hours post-initiation (e.g. that are between about ! an hour to seven hours post-initiation, between about ! an hour to seven hours postinitiation, or between about one hour to seven hours post-initiation; as an alternative (or in addition) to defining apoptotic cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0133] In embodiments (e.g. the embodiments above) that refer to detecting the presence or absence of “apoptotic cells”, exemplary cell types include cells of the connective tissue (e.g. an extracellular matrix cell, more preferably a fibroblast), a stem cell (e.g. a mesenchymal stem cell such as a bone marrow-derived mesenchymal stem cell (BMMSC)) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC).

[0134] In any embodiment described herein directed to detecting the presence or absence of an apoptotic cell of any particular cell type, it may be said that the sample (e.g. test sample) is one having or suspected of having cells of such cell type. In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), terminal mannose and terminal N- acetylglucosamine is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic connective tissue cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis.

[0135] Throughout this specification, the term “up to” preferably means “up to and including”.

[0136] Throughout this specification (in the context of any aspect or embodiment), reference to (e.g. apoptotic) connective tissue cells may preferably mean (e.g. apoptotic) extracellular matrix cells, more preferably (e.g. apoptotic) fibroblasts.

[0137] The term “post-initiation” refers to the time after initiation (or induction) of the apoptosis process, beginning with entry into the induction (aka pre -commitment) phase. By way of example, when using an apoptosis inducing agent (such as staurosporine) to initiate apoptosis, the term “post-initiation” refers a timeframe beginning from contact of a cell with such apoptosis inducing agent (whereupon the first phase of the apoptosis process is initiated).

[0138] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), terminal mannose and terminal N- acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic connective tissue cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0139] In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), terminal mannose and terminal N- acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis.

[0140] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), terminal mannose and terminal N- acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0141] In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) about 170s minute post-initiation (e.g. more preferably that are at up to (and including) 1 hour post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). Said connective tissue cells may preferably be fibroblasts.; as an alternative (or in addition) to defining apoptotic connective tissue cells by such minutes post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to about 40% (e.g. up to about 10%, 15%, 20%, 25%, 30%, or 35%) of the way through the pre-commitment (induction) phase of apoptosis.

[0142] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) about 170 minutes post-initiation (e.g. more preferably that are at up to (and including) 1 hour post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such minutes post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to about 40% (e.g. up to about 10%, 15%, 20%, 25%, 30%, or 35%) of the way through the precommitment (induction) phase of apoptosis.

[0143] In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) about 170 minute post-initiation (e.g. more preferably that are at up to (and including) 1 hour post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such minutes post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to about 40% (e.g. up to about 10%, 15%, 20%, 25%, 30%, or 35%) of the way through the pre-commitment (induction) phase of apoptosis.

[0144] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) about 170 minute post-initiation (e.g. more preferably that are at up to (and including) 1 hour post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such minutes post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to about 40% (e.g. up to about 10%, 15%, 20%, 25%, 30%, or 35%) of the way through the pre-commitment (induction) phase of apoptosis.

[0145] In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation (e.g. that are at greater than about 170 minutes post-initiation, such as between three hours and six (or seven) hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours / minutes post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at > about 15% (e.g. about 25-100%, about 30-100%, about 35-100%, about 40- 100%, about 45-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis. Said connective tissue cells may preferably be fibroblasts.

[0146] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation (e.g. that are at greater than about 170 minutes post-initiation, such as between three hours and six (or seven) hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours / minutes post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at > about 15% (e.g. about 25-100%, about 30-100%, about 35-100%, about 40-100%, about 45-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0147] In one embodiment, the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation (e.g. that are at greater than about 170 minutes post-initiation, such as between three hours and six (or seven) hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours / minutes post-initiation, it may be said that the presence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at > about 15% (e.g. about 25-100%, about 30-100%, about 35-100%, about 40- 100%, about 45-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0148] In one embodiment, the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation (e.g. that are at greater than about 170 minutes post-initiation, such as between three hours and six (or seven) hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic connective tissue cells by such hours / minutes post-initiation, it may be said that the absence has been detected of apoptotic connective tissue cells (and / or vesicles thereof) that are at > about 15% (e.g. about 25-100%, about 30-100%, about 35-100%, about 40- 100%, about 45-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0149] Said “connective tissue cells” may preferably be fibroblasts.

[0150] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose (such as a Fuc a( 1— >6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), terminal mannose and terminal N-acetylglucosamine is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about i an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0151] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 3- 100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0152] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis.

[0153] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about i an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis.

[0154] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation (e.g. that are at < about 5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 80% (e.g. about 50%, about 60% or preferably about 70%) of the way through the pre-commitment (induction) phase of apoptosis.

[0155] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation (e.g. that are at < about 5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 80% (e.g. about 50%, about 60% or preferably about 70%) of the way through the pre-commitment (induction) phase of apoptosis.

[0156] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation (e.g. that are at < about 5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 80% (e.g. about 50%, about 60% or preferably about 70%) of the way through the pre-commitment (induction) phase of apoptosis.

[0157] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation (e.g. that are at < about 5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 80% (e.g. about 50%, about 60% or preferably about 70%) of the way through the pre-commitment (induction) phase of apoptosis.

[0158] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > about 5 hours post-initiation (e.g. between

[0159] > 5 hours and up to 7 hours post-initiation, or > 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 70% (e.g. about 75-100%, about 80-100%, about 85-100%, about 90-100% or about 95-100%) of the way through the precommitment (induction) phase of apoptosis.

[0160] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > about 5 hours post-initiation (e.g. between

[0161] > 5 hours and up to 7 hours post-initiation, or > 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 70% (e.g. about 75-100%, about 80-100%, about 85-100%, about 90-100% or about 95- 100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0162] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > about 5 hours post-initiation (e.g. between

[0163] > 5 hours and up to 7 hours post-initiation, or > 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 70% (e.g. about 75-100%, about 80-100%, about 85-100%, about 90-100% or about 95-100%) of the way through the precommitment (induction) phase of apoptosis.

[0164] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > about 5 hours post-initiation (e.g. between

[0165] > 5 hours and up to 7 hours post-initiation, or > 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 70% (e.g. about 75-100%, about 80-100%, about 85-100%, about 90-100% or about 95-100%) of the way through the precommitment (induction) phase of apoptosis.

[0166] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour (e.g. between > 1 hour and up to 7 hours post initiation, or > 3 hours post-initiation, or more preferably > 3 hours postinitiation and up to 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N- acetylgalactosamine is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 10% (e.g. about 20-100%, about 30-100%, about 40-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0167] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour (e.g. between > 1 hour and up to 7 hours post initiation, or > 3 hours post-initiation, or more preferably > 3 hours postinitiation and up to 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 10% (e.g. about 20-100%, about 30-100%, about 40-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0168] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour (e.g. between > 1 hour and up to 7 hours post initiation, or > 3 hours post-initiation, or more preferably > 3 hours postinitiation and up to 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N- acetylgalactosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 10% (e.g. about 20-100%, about 30-100%, about 40-100%, or about 50-100%) of the way through the pre-commitment (induction) phase of apoptosis. In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour (e.g. between > 1 hour and up to 7 hours post initiation, or > 3 hours post-initiation, or more preferably > 3 hours postinitiation and up to 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N- acetylgalactosamine is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at > about 10% (e.g. about 20-100%, about 30-100%, about 40-100%, or about 50- 100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0169] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) about 1 hour or about > 7 hours (e.g. about 7 hours + / - 5-10 minutes) post-initiation may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 15% (e.g. up to about 10%, 12% or 14%) or that are at about 90- 100% (e.g. 95-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0170] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) about 1 hour or about > 7 hours (e.g. about 7 hours + / - 5-10 minutes) post-initiation may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 15% (e.g. up to about 10%, 12% or 14%) or that are at about 90-100% (e.g. 95-100%) of the way through the pre-commitment (induction) phase of apoptosis. In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) about 1 hour or about > 7 hours (e.g. about 7 hours + / - 5-10 minutes) post-initiation may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 15% (e.g. up to about 10%, 12% or 14%) or that are at about 90-100% (e.g. 95-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0171] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) about 1 hour or about > 7 hours (e.g. about 7 hours + / - 5-10 minutes) post-initiation may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at up to about 15% (e.g. up to about 10%, 12% or 14%) or that are at about 90-100% (e.g. 95-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0172] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation (e.g. about 1.5 to 6.5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 10-90% (e.g. about 15-85%, 20-80%, or 25-75%) of the way through the precommitment (induction) phase of apoptosis. In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation (e.g. about

[0173] 1.5 to 6.5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 10-90% (e.g. about 15-85%, 20-80%, or 25-75%) of the way through the pre-commitment (induction) phase of apoptosis.

[0174] In one embodiment, the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation (e.g. about

[0175] 1.5 to 6.5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 10-90% (e.g. about 15-85%, 20-80%, or 25-75%) of the way through the pre-commitment (induction) phase of apoptosis.

[0176] In one embodiment, the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation (e.g. about

[0177] 1.5 to 6.5 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic stem cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic stem cells (and / or vesicles thereof) that are at between about 10- 90% (e.g. about 15-85%, 20-80%, or 25-75%) of the way through the pre-commitment (induction) phase of apoptosis.

[0178] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal fucose residue (e.g. such as those capable of being bound by AAL and / or UEA-1 as described herein) is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis. Said endothelial cells may preferably be HUVEC cells.

[0179] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal fucose residue (e.g. such as those capable of being bound by AAL and / or UEA-1 as described herein) is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the precommitment (induction) phase of apoptosis.

[0180] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal fucose residue (e.g. such as those capable of being bound by AAL and / or UEA-1 as described herein) is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about i an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0181] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal fucose residue (e.g. such as those capable of being bound by AAL and / or UEA-1 as described herein) is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to six or seven hours post-initiation (e.g. that are between about ! an hour to six (or seven) hours post-initiation, between about ! an hour to six (or seven) hours post-initiation, or between about one hour to six (or seven) hours post-initiation; as an alternative (or in addition) to defining apoptotic endothelial cells by such hours postinitiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at between about 3-100% (e.g. about 5-100%, about 10-100%, or about 20-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0182] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0183] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0184] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0185] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0186] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours postinitiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60- 100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0187] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours post-initiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60- 100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0188] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours postinitiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60-100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0189] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours post-initiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours postinitiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60-100%, 70- 100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0190] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0191] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post- initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0192] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0193] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis. In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0194] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0195] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0196] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) about 3 hours post-initiation (e.g. up to an including about 2.5 hours post-initiation, more preferably up to and including about 1 hours postinitiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at up to about 50% (e.g. up to about 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, preferably up to about 15%) of the way through the pre-commitment (induction) phase of apoptosis.

[0197] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours postinitiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘lower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60- 100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0198] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours post-initiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60- 100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0199] In one embodiment, the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours postinitiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N-acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours post-initiation, it may be said that the presence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50- 100%, 60-100%, 70-100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0200] In one embodiment, the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 3 hours post-initiation (e.g. > 3 hours to about 7 hours post-initiation, > 7 hours post-initiation, more preferably 7 hours post-initiation) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); as an alternative (or in addition) to defining apoptotic endothelial cells by such hours postinitiation, it may be said that the absence has been detected of apoptotic endothelial cells (and / or vesicles thereof) that are at > about 40% (e.g. 50-100%, 60-100%, 70- 100% or 90-100%) of the way through the pre-commitment (induction) phase of apoptosis.

[0201] The more glycan types that are detected in the test sample, the greater the accuracy and reliability with which apoptotic cells and / or vesicles thereof can be identified. Therefore, the method may comprise detecting two or more, three or more, four or more, five or more, six or more, or seven or more different glycans described herein in the sample. In embodiments in which the method comprises detecting two different glycans, the glycans may comprise terminal sialic acid residues and terminal mannose residues. In embodiments in which the method comprises detecting three different glycans, the glycans may comprise terminal sialic acid residues, terminal mannose residues, and terminal N-acetylglucosamine residues. Glycans comprising terminal sialic acid residues may be alpha-linked sialic acids, such as alpha-2, 3-linked sialic acid or alpha- 2,6-linked sialic acid. Glycans comprising terminal mannose residues may be alphalinked mannose, such as alpha- 1,6-linked mannose. The method may comprise detecting the presence of a glycan comprising terminal alpha-linked sialic acid residues and terminal alpha-linked mannose residues. The method may comprise detecting one or more types of the glycans referred to herein in a sample together with other known biomarkers (e.g., proteins).

[0202] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal sialic acid residues (e.g., an alpha-linked sialic acid, such as an alpha-2, 3-linked sialic acid or an alpha-2, 6-linked sialic acid), and optionally one or more monosaccharide residues selected from the group consisting of:

[0203] (i) terminal galactose residues,

[0204] (ii) terminal mannose residues,

[0205] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0206] (iv) terminal fucose residues,

[0207] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0208] (vi) internal N-acetyllactosamine residues.

[0209] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more galactose residues, and optionally one or more monosaccharide residues selected from the group consisting of:

[0210] (i) terminal sialic acid residues,

[0211] (ii) terminal mannose residues,

[0212] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0213] (iv) terminal fucose residues,

[0214] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0215] (vi) internal N-acetyllactosamine residues. The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal mannose residues (e.g., an alpha-linked mannose, such as alpha- 1,6-linked mannose) and optionally one or more monosaccharide residues selected from the group consisting of:

[0216] (i) terminal sialic acid residues,

[0217] (ii) terminal galactose residues,

[0218] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0219] (iv) terminal fucose residues,

[0220] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0221] (vi) internal N-acetyllactosamine residues;

[0222] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal N- acetylgalactosamine (GalNAc) residues and optionally one or more monosaccharide residues selected from the group consisting of:

[0223] (i) terminal sialic acid residues,

[0224] (ii) terminal galactose residues,

[0225] (iii) terminal mannose residues,

[0226] (iv) terminal fucose residues and

[0227] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0228] (vi) internal N-acetyllactosamine residues.

[0229] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal fucose residues (e.g., an alpha linked fucose residue) and optionally one or more monosaccharide residues selected from the group consisting of:

[0230] (i) terminal sialic acid residues,

[0231] (ii) terminal galactose residues,

[0232] (iii) terminal mannose residues,

[0233] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0234] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0235] (vi) internal N-acetyllactosamine residues.

[0236] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal N- acetylglucosamine residues (e.g., Chitin, or (GlcNAc)3) and optionally one or more monosaccharide residues selected from the group consisting of:

[0237] (i) terminal sialic acid residues,

[0238] (ii) terminal galactose residues,

[0239] (iii) terminal mannose residues,

[0240] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0241] (v) terminal fucose residues and

[0242] (vi) internal N-acetyllactosamine residues.

[0243] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more internal N- acetyllactosamine residues and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0244] (i) terminal sialic acid residues,

[0245] (ii) terminal galactose residues,

[0246] (iii) terminal mannose residues,

[0247] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0248] (v) terminal fucose residues, and

[0249] (vi) terminal N-acetylglucosamine (GlcNAc) residues.

[0250] The method may comprise detecting in the sample the level of extracellular and / or extravesicular surface display of a glycan comprising one or more terminal monosaccharide residues selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1,6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, alpha linked fucose residues, such as Fuc al- 2 Gal bl -4 GlcNAc or Fuc al -2 Gal [6S] bl -4 Glc, alpha linked fucose residues such as an a( 1— >2) linked fucose linked to a galactose, a Fuc a( 1— >6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc and terminal N- acetylglucosamine residues. Alternatively or additionally, the method may comprise contacting the sample with a probe for a glycan comprising internal N- acetyllactosamine residues.

[0251] The method may comprise detecting a glycan comprising terminal monosaccharide residues that are indicative of apoptosis using a probe (e.g., a lectin, an antibody, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition or a cell). The probe may be specific and / or selective for the glycan(s). The probe may most preferably be a lectin (e.g. a lectin selected from the group consisting of: AAL, GNA, MAL-II, NPL, UEA-I, SNA, STL, SJA and PNA). The probe may be referred to as a “binding partner”; following challenge of the test sample with the probe / binding partner, methods of the invention may comprise detecting a level of probe / binding partner-glycan complex.

[0252] A glycan detected by methods of the invention may be referred to by reference to a complementary lectin having affinity for the glycan. For example, the one or more residues (of the glycan) described herein may be capable of being bound by a lectin selected from the group consisting of: AAL, GNA, MAL-II, NPL, UEA-I, SNA, STL, SJA and PNA.

[0253] The skilled person understands which glycans said lectins bind to, and would thus understand the what the “one or more residues (of the glycan)” is when described with reference to the lectin it is bound by. However, for the avoidance of doubt, and to summarise what was outlined above: terminal fucose “an a(1^2) linked fucose linked to a galactose” may be referred to as a terminal glycan residue capable of being bound by Ulex Europaeus Agglutinin I (UEA- i); terminal fucose such as “a Fuc a( I— >6) GlcNAc, a Fuc a( I— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1 — >6) GlcNAc)” may be referred to as a terminal glycan residue capable of being bound by AAL; terminal sialic acid that is an “alpha-2, 3-linked sialic acid” (preferably a terminal NeuAc a2-3 Gal bl -3 GalNAc residue) may be referred to as a terminal glycan residue capable of being bound by MAL-II; terminal sialic acid that is an alpha-2, 6-linked sialic acid (preferably a terminal NeuAc a2-6 Gal bl -4 GlcNAc residue) may be referred to as a terminal glycan residue capable of being bound by SNA; terminal N-acetylglucosamine (preferably a P(l,4)-linked GlcNAc) and / or an oligomer thereof (e.g. Chitin and / or (GlcNAc)3) may preferably be referred to as a terminal glycan residue capable of being bound by STL; terminal mannose that is an “alpha- 1,6-linked mannose” (preferably alpha- 1,6-linked Man3GlcNAc2 and / or alpha- 1,6-linked Man5GlcNAc2) may be referred to as a terminal glycan residue capable of being bound by NPL; terminal mannose that is an “alpha-1, 3-linked mannose” (preferably alpha- 1,3 -linked Man3GlcNAc2) may be referred to as a terminal glycan residue capable of being bound by GNA; terminal galactose “Fuc al -2 (Gal bl -3 GalNAc al -3) Gal bl -4 GlcNAc” may be referred to as a terminal glycan residue capable of being bound by PNA; terminal galactose “Fuc al -3 (Fuc al -2 (Gal al -3) Gal bl -4) GlcNAc” may be referred to as a terminal glycan residue capable of being bound by SJA; terminal N-acetylgalactosamine (preferably GalNAc b 1 -4 GlcNAc b 1 -3 GalNAc b 1 -4 GlcNAc) may be referred to as a terminal glycan residue capable of being bound by SJA.

[0254] Thus, the method may comprise detecting, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising terminal monosaccharide residues and internal residues capable of being bound by a lectin selected from the group consisting of: AAL, GNA, MAL-II, NPL, UEA-I, SNA, STL, SJA and PNA, comparing this level of display with a reference level for the glycan(s) in a reference sample that does not comprise apoptotic cells and / or vesicles thereof, wherein a difference in the level of display of the glycan detected in the sample relative to the level of display of the glycan the reference sample is indicative that the test sample comprises apoptotic cells and / or vesicles thereof.

[0255] The inventors have identified that a number of glycan molecules, defined by their terminal monosaccharide residues, and / or internal N-acetyllactosamine residues, which surprisingly change extracellular and / or extravesicular surface display levels when a cell initiates apoptosis. For example, a number of glycans comprising specific terminal monosaccharide residues and / or specific internal residues exhibit a lower level of display in apoptotic cells and / or vesicles thereof compared to the level of display in non-apoptotic cells and / or vesicles thereof. Hence the level of display of the glycan referred to herein may act as a biomarker for apoptotic cells and / or vesicles thereof. The difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be a higher level of display or a lower level of display. A higher level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be indicative that the sample comprises one or more apoptotic cells and / or vesicles thereof. A lower level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be indicative that the sample comprises one or more apoptotic cells and / or vesicles thereof.

[0256] In some embodiments, the method may comprise detecting the level of extracellular and / or extravesicular surface display of a plurality of the glycans disclosed herein. In such embodiments, the pattern of display, where glycans are displayed at either higher or lower (for example significantly higher or significantly lower) levels in a test sample compared to a reference sample may be referred to as the “glycan signature ” . The glycan signature represents a useful and robust marker for the presence or absence of apoptotic cells and / or vesicles thereof. Thus, a glycan signature may be used to indicate whether a sample comprises apoptotic cells and / or vesicles thereof.

[0257] The invention may comprise detecting the level of extracellular and / or extravesicular surface display of a glycan in a sample after the initiation of apoptosis, this may be in the first 10 minutes, first 30 minutes, first hour, or about an hour or more, after the initiation of apoptosis. However, the glycan signature per se may change with time (although the diagnostic potential of the glycan signature may not change). There are at least two key timepoints at which a glycan signature may be used as part of the invention: (i) after the initiation of apoptosis up to about 3 hours from initiation, for example about 1 hour or earlier to about 3 hours after the initiation of apoptosis; and (ii) about 4 hours to about 6 hours or more after the initiation of apoptosis. Thus, the method according to the invention may comprise detecting a glycan after the initiation of apoptosis, at about 1 hour to about 3 hours after the initiation of apoptosis. The method according to the invention may comprise detecting a glycan at about 4 hours to about 6 hours or more after the initiation of apoptosis.

[0258] As discussed in the Examples, at any point after the initiation of apoptosis, for example about 1 hour or earlier to about 3 hours after the initiation of apoptosis, a higher level of extracellular and / or extravesicular surface display of a glycan comprising terminal monosaccharide residues bound by lectins, for example SNA (e.g., terminal alpha-2, 6- linked sialic acid residues) or SJA (e.g., terminal galactose or N-acetylgalactosamine residues), can be detected compared to the level of extracellular and / or extravesicular surface display of the glycan in apoptotic cells and / or vesicles thereof. Thus, the method may comprise detecting, in the sample, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 1 hour to 3 hours after the initiation of apoptosis, the level of extracellular and / or extravesicular surface display of a glycan comprising terminal alpha-2, 6-linked sialic acid residues, wherein detection of a higher level of extracellular and / or extravesicular surface display of the glycan compared to a reference level of display of the glycan detected in a reference sample is indicative that the sample contains apoptotic cells and / or vesicles thereof.

[0259] Also discussed in the Examples, 1 hour or earlier to 3 hours after the initiation of apoptosis, a lower level of extracellular and / or extravesicular surface display of a glycan comprising terminal monosaccharide residues bound by the lectins, AAL, GNA, MAL- II, NPL, UEA-I, and STL, can be detected compared to the level of extracellular and / or extravesicular surface display of the glycan in non-apoptotic cells and / or vesicles thereof. Thus, the method may comprise detecting, in the sample, about 1 hour or earlier to 3 hours after the initiation of apoptosis, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more terminal residues selected from the group consisting of:

[0260] (i) terminal sialic acid residues, such as alpha-2, 3-linked sialic acid residues and / or alpha-2, 6-linked sialic acid residues,

[0261] (ii) terminal galactose residues,

[0262] (iii) terminal mannose residues, such as alpha- 1, 6-linked mannose residues and / or alpha-1, 3-linked mannose residues,

[0263] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0264] (v) terminal fucose residues,

[0265] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0266] (vii) internal N-acetyllactosamine residues; wherein detection of a lower level of extracellular and / or extravesicular surface display of the glycan compared to a reference level of display of the glycan detected in a reference sample is indicative that the sample comprises apoptotic cells and / or vesicles thereof.

[0267] Also disclosed herein is a kit that may be used to perform the method according to the invention. According to a second aspect of the invention, there is provided a kit for detecting apoptotic cells and / or vesicles thereof in a sample of cells, the kit comprising: a probe for a glycan; wherein the glycan comprises one or more residues selected from the group consisting of:

[0268] (i) terminal sialic acid residues,

[0269] (ii) terminal galactose residues,

[0270] (iii) terminal mannose residues,

[0271] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0272] (v) terminal fucose residues,

[0273] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0274] (vii) internal N-acetyllactosamine residues; preferably wherein the probe of the kit is used to determine, in a test sample, the level of display of one or more of the glycans, wherein a difference in the level of display of one or more of the glycans in the test sample relative to a level of display of one or more of the glycans in a reference sample, indicates the presence of apoptotic cells and / or vesicles thereof in the test sample, optionally this can be used in the diagnosis of a apoptotic-related disease. The reference sample may not contain apoptotic cells and / or vesicles thereof.

[0275] In one embodiment, the kit comprises: a probe for a glycan; wherein the glycan comprises monosaccharide residues selected from the group consisting of:

[0276] (i) terminal sialic acid residues, such as alpha-2, 3-linked sialic acid residues and / or alpha-2, 6-linked sialic acid residues,

[0277] (ii) terminal galactose residues,

[0278] (iii) terminal mannose residues, such as alpha- 1, 6-linked mannose residues and / or alpha-1, 3-linked mannose residues,

[0279] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0280] (v) terminal fucose residues,

[0281] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0282] (vii) internal N-acetyllactosamine residues; preferably wherein the probe of the kit is used to detect, in a test sample, the level of display of one or more of the glycans. When levels of the glycans in the test sample are compared to the levels in a reference sample, wherein the reference sample does not contain apoptotic cells and / or vesicles thereof, if a difference is observed it may then be concluded that the test sample contains apoptotic cells and / or vesicles thereof, this may help diagnosis of an apoptotic-related disease. The group from which terminal monosaccharide residues may be selected from optionally further consists of: NeuAc a2-3 Gal bl -3 GalNAc, NeuAc a2-6 Gal bl -4 GlcNAc, Fuc al-2( Gal bl- 3 GalNAc al-3) Gal bl-4 GlcNAc, Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc, GalNAc bl-4 GlcNAc bl -3 GalNAc bl-4 GlcNAc, Man3GlcNAc2, Man5GlcNAc2, an a(1^2) linked fucose linked to a galactose (e.g. Fuc al-2 Gal bl-4 GlcNAc), and Fuc al-2 Gal [6S] bl-4 Glc. In addition or alternatively, the probe may be for internal N-acetyllactosamine (e.g. LacNAc) residues such as Chitin or (GlcNAc)3 (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds). In addition or alternatively, the probe may be for internal N- acetyllactosamine (e.g. LacNAc) residues such as poly-LacNAc (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by - 1,4 bonds).

[0283] A kit of the invention may comprise (e.g. as a physical feature / component of the kit) technical instructions to use the kit to detect the presence or absence of apoptotic cells (and / or vesicles thereof) by a method described herein.

[0284] The probe may be for enriching apoptotic cells and / or vesicles thereof.

[0285] It is particularly preferred that a kit described herein may comprise a further agent for detecting a further biomarker (e.g. a protein biomarker) of apoptosis.

[0286] The kit may comprise an imaging agent. Thus, an imaging agent may be conjugated or attached to the probe. The imaging agent may be a fluorophore, a radioisotope or a dye (preferably a fluorophore). The kit may comprise a conjugating agent. The conjugating agent may be used to conjugate the probe with the imaging agent. The kit may comprise a probe, or probes, conjugated to an imaging agent (or imaging agents).

[0287] A kit described herein may comprise a probe for terminal fucose, preferably a probe for a terminal a( 1 ^2) linked fucose linked to a galactose (e.g. an alpha-glycosidic linked fucose). Said probe for terminal fucose may preferably be Ulex Europaeus Agglutinin I (UEA-I), optionally conjugated to an imaging agent. Such kit (comprising said probe for terminal fucose) may be particularly suitable for detecting the presence or absence of apoptotic endothelial cells (e.g HUVEC cells) in a sample, e.g. via a method described herein.

[0288] Additionally or alternatively, a kit described herein may comprise a probe for a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1 — >6) GlcNAc), e.g. a terminal fucose. Said probe for a Fuc a( 1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1 — >6) GlcNAc) may preferably be Aleuria Aurantia Lectin (AAL), optionally conjugated to an imaging agent. Such kit (comprising said probe for a Fuc a( 1 — >6) GlcNAc, a Fuc a( 1 ^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1^4) GlcNAc (preferably a Fuc a( 1^6) GlcNAc) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC).

[0289] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-2, 3-linked sialic acid, preferably a probe for terminal NeuAc a2-3 Gal bl-

[0290] 3 GalNAc residue. Said probe for terminal sialic acid may preferably be Maackia Amurensis Lectin II (MAL-II), optionally conjugated to an imaging agent. Such kit (comprising said probe for an alpha-2, 3-linked sialic acid) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC).

[0291] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-2, 6-linked sialic acid, preferably a probe for a terminal NeuAc a2-6 Gal bl-

[0292] 4 GlcNAc residue. Said probe for a terminal alpha-2, 6-linked sialic acid may preferably be Sambucus Nigra Lectin (SNA), optionally conjugated to an imaging agent. Such kit (comprising said probe for a terminal alpha-2, 6-linked sialic acid) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC). Additionally or alternatively, a kit described herein may comprise a probe for a terminal N-acetylglucosamine (e.g. as monomer and / or oligomer), preferably a probe for ( 1 ,4)- linked GlcNAc. Said probe for a terminal N-acetylglucosamine may preferably be Solanum Tuberosum Lectin (STL), optionally conjugated to an imaging agent. Such kit (comprising said probe for a terminal N-acetylglucosamine) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC).

[0293] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha- 1,6-linked mannose, preferably a probe for alpha- 1,6-linked Man3GlcNAc2 and / or alpha- 1,6-linked Man5GlcNAc2. Said probe for a terminal alpha- 1,6-linked mannose may preferably be Narcissus Pseudonarcissus Lectin (NPL), optionally conjugated to an imaging agent. Such kit (comprising said probe for a terminal alpha- 1,6-linked mannose) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC).

[0294] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-1, 3-linked mannose, preferably a probe for alpha-1, 3-linked mannose Man3GlcNAc2. Said probe for a terminal alpha-1, 3-linked mannose may preferably be Galanthus Nivalis Lectin (GNA), optionally conjugated to an imaging agent. Such kit (comprising said probe for a terminal alpha-1, 3-linked mannose) may be particularly suitable for detecting the presence or absence of an apoptotic connective tissue cell (e.g. fibroblast), an apoptotic stem cell (e.g. mesenchymal stem cell) and / or an apoptotic endothelial cell (e.g. human umbilical vein endothelial cell, HUVEC).

[0295] Additionally or alternatively, a kit described herein may comprise a probe for a terminal galactose, preferably a probe for a galactosyl (P-1,3) N-acetylgalactosamine, such as Fuc al-2( Gal b 1-3 GalNAc al-3) Gal bl-4 GlcNAc. Said probe for a terminal galactose may preferably be Peanut Agglutinin (PNA), optionally conjugated to an imaging agent. Such kit (comprising said probe for a terminal galactose) may be particularly suitable for detecting the presence or absence of an apoptotic stem cell (e.g. mesenchymal stem cell). The kit may further comprise a control sample and / or a reference sample. The kit may further comprise a positive control and / or a negative control sample. The positive control may be a sample that comprises apoptotic cells and / or vesicles thereof. The negative control may be a sample that does not comprise or contain apoptotic cells and / or vesicles thereof. The reference sample may be a sample of cells. The reference sample may be one of the control samples or may be a different sample. The reference sample may be a sample that comprises cells that do not display any of the glycans referred to herein, or low, undetectable levels of the glycan referred to herein. The reference sample may comprise non-apoptotic cells and / or vesicles thereof.

[0296] The method according to the first aspect of the invention may be used to enrich non- apoptotic or apoptotic cells in a sample.

[0297] Thus, according to a third aspect, there is provided a method of enriching apoptotic or non-apoptotic cells in a sample, the method comprising: contacting the sample, which comprises a mixture of non-apoptotic cells (and / or vesicles thereof) and apoptotic cells (and / or vesicles thereof), with a probe for a glycan; and enriching the non-apoptotic (and / or vesicles thereof), or the apoptotic cells (and / or vesicles thereof), using the probe.

[0298] Exemplary cell types include cells of the connective tissue (e.g. a fibroblast), a stem cell (e.g. a mesenchymal stem cell, such as a bone marrow mesenchymal stem cell) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC).

[0299] In one embodiment, the method comprises: contacting the sample, which comprises a mixture of non-apoptotic cells and / or vesicles thereof and apoptotic cells and / or vesicles thereof, with a probe for a glycan; and enriching for the non-apoptotic cells and / or vesicles thereof using the probe, wherein the probe recognises one or more types of residues of the glycan selected from the group consisting of:

[0300] (i) terminal sialic acid residues, (ii) terminal galactose residues,

[0301] (iii) terminal mannose residues,

[0302] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0303] (v) terminal fucose residues,

[0304] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0305] (vii) internal N-acetyllactosamine residues.

[0306] The method may comprise contacting the sample with a probe for a glycan comprising terminal monosaccharide residues selected from the group consisting of terminal alpha- 2,3-linked sialic acid residues, terminal alpha-2, 6-linked sialic acid residues, alpha-1, 6- linked mannose residues, alpha-1, 3-linked mannose residues, NeuAc a2-3 Gal bl-

[0307] 3 GalNAc, NeuAc a2-6 Gal bl -4 GlcNAc, Fuc al-2( Gal bl -3 GalNAc al -3) Gal bl-

[0308] 4 GlcNAc, fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc, GalNAc bl-4 GlcNAc bl-

[0309] 3 GalNAc bl-4 GlcNAc, Man3GlcNAc2, Man5GlcNAc2, an a( l ^2) linked fucose linked to a galactose (e.g. Fuc al-2 Gal bl-4 GlcNAc), and Fuc al-2 Gal [6S] bl-

[0310] 4 Glc. In addition or alternatively, the probe may be for internal N-acetyllactosamine (e.g. LacNAc) residues such as Chitin, (GlcNAc)3 (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds). In addition or alternatively, the probe may be for internal N-acetyllactosamine (e.g. LacNAc) residues such as poly-LacNAc (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds).

[0311] It will be appreciated that the method according to the invention may be used to remove apoptotic or non-apoptotic cells and / or vesicles thereof from the sample. The sample may be from a subject. The sample may be an in vivo, in vitro or ex vivo sample (preferably in vitro or ex vivo). In embodiments in which the invention is performed in vivo, the method may be used to remove apoptotic cells and / or vesicles thereof, or non- apoptotic cells and / or vesicles thereof, from a subject. In embodiments in which the invention is performed ex vivo, the method may be used to treat a disease or a condition in a subject.

[0312] The method according to the invention may be repeated multiple times on a single sample in order to improve the purity of the sample. Thus, the method according to the invention may be performed twice, three times, four times or more on a single test sample. It will be appreciated that (performing) the contacting step leads to the probe being bound to the glycan(s) displayed on the apoptotic cells and / or vesicles thereof, or the probe being bound to the glycan(s) displayed on the non-apoptotic cells and / or vesicles thereof. The probe may be for a glycan referred to herein. The probe may be specific and / or selective for the glycan.

[0313] The method according to the invention may further comprise the step of providing a sample (prior to the contacting step). The step of providing the sample may comprise providing the cells in culture (prior to the contacting step). Thus, the method according to the invention may or may not comprise extracting the cells from a subject. Thus, the method according to the invention may comprise providing cells that have already been obtained from a subject.

[0314] The enriching step of the invention may comprise using the probe to detect and / or separate and / or isolate either non-apoptotic cells and / or vesicles thereof in the test sample or apoptotic cells and / or vesicles thereof in the sample. Thus, the enriching step may comprise using the probe to separate / isolate non-apoptotic cells and / or vesicles thereof, from apoptotic cells and / or vesicles thereof; or separate / isolate apoptotic cells and / or vesicles thereof from non-apoptotic cells and / or vesicles thereof. Thus, enriching may comprise contacting the sample with one or more probes that bind to the glycans referred to herein and then separating / isolating either non-apoptotic cells and / or vesicles thereof, or apoptotic cells and / or vesicles thereof, using the probe. Separation / isolation may be performed using bio-physical separation techniques known in the art, including for example, flow cytometry sorting, magnetic bead separation, separation columns, a bioreactor, or other flow-based technique. Enrichment may be performed using magnetic bead separation. Enrichment may be performed on live / intact cells.

[0315] Enrichment of non-apoptotic cells and / or vesicles thereof may be performed by a probe for a glycan binding to and allowing removal of apoptotic cells and / or vesicles thereof (thus leaving behind unbound non-apoptotic cells and / or vesicles thereof that do not display a glycan, e.g., a glycan referred to herein). Alternatively, enrichment of non- apoptotic cells may be performed using a probe that binds to and allows the isolation of non-apoptotic cells and / or vesicles thereof. The probe may be a lectin. In one embodiment, the lectin may be a selection of one or more of AAL, GNA, MAL-II, NPL, UEA-I, SNA, SJA, PNA and STL. The lectin may be SNA. The probe (e.g., the lectin) may be used after the initiation of apoptosis, for example within an hour of apoptosis initiation, about 10 minutes, about 30 minutes, about 1 hour, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, or 1 hour to about 2 hours, after the initiation of apoptosis to enrich non-apoptotic cells. The enrichment of non-apoptotic cells and / or vesicles thereof may be performed in at least two steps. One of the at least two steps (e.g., the first or the second step) may comprise enriching the non-apoptotic cells and / or vesicles thereof using SNA. The other of the at least two steps (e.g., the first or the second step) may comprise enriching the non- apoptotic cells and / or vesicles thereof using AAL, GNA, MAL-II, NPL, UEA-I, PNA, SJA and / or STL.

[0316] Similarly, enrichment of apoptotic cells may be performed by a probe for a glycan binding to and allowing the removal of non-apoptotic cells and / or vesicles thereof (thus leaving behind unbound apoptotic cells and / or vesicles thereof). Alternatively, enrichment of apoptotic cells and / or vesicles thereof may be performed by a probe binding to and allowing the removal of apoptotic cells and / or vesicles thereof.

[0317] The probe may be a lectin. In one embodiment, the lectin may be a selection of one or more of AAL, GNA, MAL-II, NPL, UEA-I, SNA, PNA, SJA and STL. The probe (e.g, the lectin) may be used after the initiation of apoptosis, for example within an hour of apoptosis initiation, about 10 minutes, about 30 minutes, about 1 hour, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, or 1 hour to about 2 hours, after the initiation of apoptosis to enrich apoptotic cells and / or vesicles thereof. The enrichment of apoptotic cells and / or vesicles thereof may be performed in at least two steps. One of the at least two steps (e.g., the first or the second step) may comprise enriching the apoptotic cells and / or vesicles thereof using AAL, GNA, MAL-II, NPL, UEA-I, SNA, PNA, SJA and / or STL. The other of the at least two steps (e.g., the first or the second step) may comprise enriching the apoptotic cells and / or vesicles thereof using SNA.

[0318] The choice of an appropriate probe depends on whether the method comprises (i) binding (e.g. immobilising or isolating) apoptotic cells and / or vesicles thereof via binding of the probe to the same, or (ii) binding (e.g. immobilising or isolating) non- apoptotic cells and / or vesicles thereof via binding of the probe to the same. Suitable probes for either scenario are discussed below.

[0319] For example, any probe (e.g. lectin) described herein may be for binding to (e.g. preferentially binding to, or specifically binding to) non-apoptotic cells, as at least certain timepoints post-initiation of apoptosis are associated with ‘lower’ levels (vs non-apoptotic reference) of any given glycan. That being said, it may be advantageous to use particular probes depending on cell type and apoptosis stage, as outlined below.

[0320] Reference to a probe for “binding to non-apoptotic cells (and / or vesicles thereof)” preferably means that the probe preferentially binds to non-apoptotic cells, with “preferentially binds” meaning that the probe binds a higher number of (i) non-apoptotic cells (and / or vesicles thereof) than (ii) apoptotic cells (and / or vesicles thereof) in a mixed population comprising both (i) and (ii). Similarly, reference to a probe for “binding to apoptotic cells (and / or vesicles thereof)” preferably means that the probe preferentially binds to apoptotic cells, with “preferentially binds” meaning that the probe binds a higher number of (i) apoptotic cells (and / or vesicles thereof) than (ii) non- apoptotic cells (and / or vesicles thereof) in a mixed population comprising both (i) and (ii). Reference to said term “higher number” may mean that the probe binds at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% more of (i) than (ii).

[0321] A preferred probe for binding to non-apoptotic cells (and / or vesicles thereof) may be a probe for binding extracellular (and / or extravesicular) displayed terminal fucose, such as a terminal alpha- 1,6-linked mannose and / or a terminal alpha-1, 3-linked mannose. As such, a probe for binding to non-apoptotic cells may be NPL and / or GNA. Advantageously, extracellular and / or extravesicular surface display of these glycans have been demonstrated to be consistently Tower’ (vs non-apoptotic reference) following initiation of apoptosis across multiple cell types, and multiple time points post-initiation of apoptosis. Said probe (for terminal fucose) may be for binding (e.g. preferentially binding) non-apoptotic cells over apoptotic cells that are at up to six hours post-initiation.

[0322] A probe for binding non-apoptotic connective tissue cells (and / or vesicles thereof) may be for binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3-linked sialic acid (e.g. with MAL-II), terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc) (e.g. with AAL), terminal mannose such as a terminal alpha-1, 6- linked mannose and / or a terminal alpha-1, 3-linked mannose (e.g. with GNA and / or NPL) and / or terminal N-acetylglucosamine (e.g. with STL). As such, a probe for binding to non-apoptotic connective tissue cells (and / or vesicles thereof) may be MAL- II, AAL, GNA, NPL and / or STL. Said probes may be for binding (e.g. preferentially binding) non-apoptotic connective tissue cells over apoptotic connective tissue cells that are at up to six hours post-initiation.

[0323] A probe for binding (e.g. preferentially binding) non-apoptotic connective tissue cells (and / or vesicles thereof) over apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal alpha-2, 6-linked sialic acid, such as probe SNA.

[0324] A probe for binding non-apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be for binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3-linked sialic acid (e.g. with MAL-II), terminal fucose (such as a Fuc a(1^6) GlcNAc, a Fuc a( I— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( I— >6) GlcNAc) (e.g. with AAL), terminal mannose such as a terminal alpha- 1, 6-linked mannose and / or a terminal alpha-1, 3-linked mannose (e.g. with GNA and / or NPL) and / or terminal N-acetylglucosamine (e.g. with STL). As such, a probe for binding to non-apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) may be MAL-II, AAL, GNA, NPL and / or STL. Said probes may be for binding (e.g. preferentially binding) non-apoptotic stem cells, such as mesenchymal stem cells, over apoptotic stem cells, such as mesenchymal stem cells, that are at up to six hours post-initiation.

[0325] A probe for binding non-apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) over apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal galactose, such as probe PNA.

[0326] A probe for binding non-apoptotic endothelial cells (and / or vesicles thereof) over apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation may be for binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3- linked sialic acid (e.g. with probe MAL-II) and / or terminal N-acetylglucosamine (e.g. with probe STL).

[0327] A probe for binding apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal alpha-2, 6-linked sialic acid, such as probe SNA.

[0328] A probe for binding apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at > 3 hours post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal N-acetylgalactosamine, such as probe SJA.

[0329] A probe for binding apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal galactose, such as probe PNA.

[0330] A probe for binding apoptotic endothelial cells (and / or vesicles thereof) (e.g. that are up to an including 7 hours post-initiation) may be for binding extracellular (and / or extravesicular) displayed terminal fucose (e.g. such as those capable of being bound by AAL and / or UEA-1 as described herein), such as probe AAL and / or UEA-1.

[0331] A probe for binding apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation may be for binding extracellular (and / or extravesicular) displayed terminal alpha-2, 3-linked sialic acid, such as probe MAL-II.

[0332] A probe for binding apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation may be for binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 6-linked sialic acid (e.g. with probe SNA) and / or terminal N-acetylglucosamine (e.g. with probe STL).

[0333] The skilled person will appreciate that although the level of display of the glycans generally reduces with time, probes (e.g., lectin or antibody) specific for these glycans are still capable of specifically binding to the glycan as long as they are used selectively (i.e., at the correct concentration). The concentration of the probe to be used will vary depending on a number of factors, including the type of cell to be bound / enriched. The skilled person would appreciate that, when using a lectin probe, the lectin concentration used during staining would be one that provides staining without causing significant agglutination / cell death / saturation. Lectin titration to appropriate concentrations for ‘staining’ is well within standard abilities of the skilled person.

[0334] The method according to the first aspect of the invention may be used to detect non- apoptotic cells and / or vesicles thereof or apoptotic cells and / or vesicles thereof to be enriched in a sample.

[0335] Hence, according to fourth aspect, there is provided a kit for enriching or removing non-apoptotic cells and / or vesicles thereof, or apoptotic cells and / or vesicles thereof, in a sample, the kit comprising: a separation means for separating non-apoptotic cells and / or vesicles thereof from apoptotic cells and / or vesicles thereof; or a separation means for separating apoptotic cells and / or vesicles thereof from non-apoptotic cells and / or vesicles thereof; and a probe for a glycan; wherein the probe is directed to glycans comprising one or more residues selected from the group consisting of:

[0336] (i) terminal sialic acid residues,

[0337] (ii) terminal galactose residues,

[0338] (iii) terminal mannose residues,

[0339] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0340] (v) terminal fucose residues,

[0341] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0342] (vii) internal N-acetyllactosamine residues; wherein the probe binds to apoptotic cells and / or vesicles thereof that extracellularly and / or extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (non- apoptotic) cells from bound (apoptotic) cells and / or vesicles thereof, thereby enriching the non-apoptotic cells and / or vesicles thereof within the test sample, or wherein the probe binds to non-apoptotic cells and / or vesicles thereof that extracellularly and / or extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii) and the separation means uses the probe to separate unbound (apoptotic) cells and / or vesicles thereof from bound (non- apoptotic) cells and / or vesicles thereof, thereby enriching the apoptotic cells and / or vesicles thereof within the test sample.

[0343] In another embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of: alpha-2, 3-linked sialic acid residues, alpha-2, 6- linked sialic acid residues, alpha linked mannose residues, alpha- 1,6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, N-acetylglucosamine residues and fucose residues such as alpha linked fucose residues. Alternatively, or additionally, the method may comprise contacting the sample with a probe for a glycan comprising internal N-acetyllactosamine residues.

[0344] The separation means of the kit may be used with a probe to separate cells bound or capable of being bound by the probe (e.g., apoptotic cells) from cells unbound or incapable of being bound by the probe (e.g., non-apoptotic cells). For example, the separation means may be magnetic separation beads.

[0345] The skilled person will appreciate these magnetic beads may be conjugated to the probe by any means known in the art. The separation means may be conjugated to the probe to create a conjugated probe. The kit may therefore comprise a means for conjugating the separation means to the probe, so as to create a conjugated probe. The kit may comprise a conjugating agent. The conjugating agent may be used to conjugate the probe with the magnetic beads. The kit may comprise a conjugated probe, or probes, conjugated to one or more separation means.

[0346] The probe may be for enriching non-apoptotic cells and / or vesicles thereof. The probe may be for binding a glycan displayed on an extracellular and / or extravesicular surface (of apoptotic cells and / or vesicles thereof). The probe may be for enriching apoptotic cells and / or vesicles thereof. The probe may be for binding a glycan displayed on an extracellular surface and / or extravesicular (of non-apoptotic cells and / or vesicles thereof). The probe may be for a glycan referred to herein. The probe may be a lectin referred to herein (e.g., AAL, GNA, MAL-II, NPL, SNA, UEA-I, PNA, SJA and / or STL). The probe (e.g., the lectin) may be for a glycan referred to herein. The kit may further comprise a control sample. The control sample may be a sample of cells. The kit may further comprise a positive control and / or a negative control sample. The positive control may be a sample that comprises apoptotic cells and / or vesicles thereof; or the positive control may be a sample that comprises non-apoptotic cells and / or vesicles thereof. The negative control (or reference sample) may be a sample that does not comprise or contain apoptotic cells and / or vesicles thereof (e.g., thus comprises non-apoptotic cells and / or vesicles thereof). The negative control (or reference sample) may be a sample that does not comprise or contain non-apoptotic cells and / or vesicles thereof (e.g., thus comprises apoptotic cells and / or vesicles thereof). The kit may further comprise, or be intended to be use with, a reference sample. The reference sample may be a sample that comprise cells or vesicles thereof that do not display any of the glycans referred to herein, or low, undetectable levels of the glycans referred to herein.

[0347] According to another aspect, there is provided a method of treating or preventing a disease or disorder in a subject, the method comprising: enriching non-apoptotic or apoptotic cells and / or vesicles thereof in a sample comprising a mixture of apoptotic and non-apoptotic cells and / or vesicles therefor using a method or a kit according to the invention; and administering the enriched sample of non-apoptotic or apoptotic cells and / or vesicles thereof to the subject to treat the disease or disorder.

[0348] The sample may be an in vitro, an in vivo or an ex vivo sample. The disease or disorder may be any disease or disorder referred to herein. The sample may be a sample of heterologous cells or a sample of autologous cells. The disease or disorder referred to herein may be a cardiovascular disease, a neurodegenerative disease or other diseases associated with cell apoptosis.

[0349] According to another aspect, there is provided an apoptotic cell probe-agent conjugate, the conjugate comprising: a probe for targeting and / or binding to a glycan comprising one or more residues selected from the group consisting of:

[0350] (i) terminal sialic acid residues,

[0351] (ii) terminal galactose residues,

[0352] (iii) terminal mannose residues,

[0353] (iv) terminal N-acetylgalactosamine (GalNAc) residues, (v) terminal fucose residues,

[0354] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0355] (vii) internal N-acetyllactosamine residues, conjugated to an agent.

[0356] The probe may be any probe referred to herein (e.g., a lectin, an antibody, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition or a cell). The glycan may comprise a terminal monosaccharide residue selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylglucosamine residues, N- acetylgalactosamine residues and fucose residues such as alpha linked fucose residues. Alternatively or additionally, the method may comprise contacting the sample with a probe for a glycan comprising internal N-acetyllactosamine residues.

[0357] The probe referred to herein may be bispecific.

[0358] The conjugate may be used to visualise an apoptotic cell and / or vesicle thereof, or tag it for detection, elimination or enrichment. In embodiments in which the conjugate is used to visualise an apoptotic cell or tag it for detection, the agent may be an imaging agent. The imaging agent may be a fluorophore, a radioisotope, a dye, or another agent used to image cells.

[0359] According to another aspect, there is provided a method of visualizing apoptotic cells and / or vesicles thereof in a subject, the method comprising: administering a probe to the subject, wherein the probe comprises an imaging agent; and detecting the imaging agent in the subject in order to visualise apoptotic cells and / or vesicles thereof in the subject.

[0360] The probe may be a probe referred to herein, such as a lectin. The probe (e.g., lectin) may be monovalent. The probe may be specific and / or selective. The probe may be attached or conjugated to the imaging agent. The imaging agent may be used to detect the location of the probe once it has been administered to the subject. The imaging agent may be a fluorophore, a radioisotope or a dye. Where the imaging agent is a dye, the dye may be detected using a whole-body imaging scan, such as a magnetic resonance imaging (MRI) scan. Thus, detecting may be performed using a whole body imagine system, such as an MRI scan. Advantageously, the method can be used to monitor apoptosis in vivo, in real time.

[0361] According to another aspect, there is provided a method of diagnosing an apoptosis- related disease or disorder in a subject, the method comprising: providing a sample obtained from a subject; detecting, in the sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0362] (i) terminal sialic acid residues,

[0363] (ii) terminal galactose residues,

[0364] (iii) terminal mannose residues,

[0365] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0366] (v) terminal fucose residues,

[0367] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0368] (vii) internal N-acetyllactosamine residues; wherein detection, in the sample, of a glycan comprising residues selected from the group consisting of (i) to (vii) is indicative that the subject has an apoptosis-related disease or disorder.

[0369] In an embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of: alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, terminal N-acetylglucosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan comprises internal N-acetyllactosamine residues.

[0370] Apoptosis-related diseases or disorders may include but are not limited to cardiovascular diseases such as myocardial infarction, neurodegenerative diseases such as Alzheimer’s, cancer, autoimmunity, liver disease, type 1 diabetes, retinal degeneration, HIV / AIDS and others.

[0371] The invention may further provide a method of treating an apoptosis-related disease or disorder by administering an enriched sample of either non-apoptotic or apoptotic cells and / or vesicles thereof to the subject to treat the disease or disorder and / or the step of administering a probe-agent conjugate according to the invention to the subject to treat the disease or disorder. The enriched samples may be prepared using a method according to the invention.

[0372] The term “terminal” may refer to the last five, last four, last three or last two residues of the glycan. The term “terminal” may refer to the last or final residue of the glycan. The term “terminal” may refer to the residues located at the terminus situated furthest from the relevant cell or vesicle. The term "terminal" may refer to the last or final residue of the glycan, which is located at the terminus situated furthest from the relevant cell or vesicle.

[0373] The term “internal” for example “internal N-acetyllactosamine residues” refers to residues that lie underneath the terminal residue.

[0374] The probe is capable of binding to the glycan(s) displayed on the surface of apoptotic cells and / or vesicles thereof, the probe may bind to the terminal residues thereof. The probe may be specific or selective for one or more of the glycans referred to herein, or the terminal residues thereof. The probe may be contacted with a sample of cells and / or vesicles thereof to produce cells and / or vesicles thereof bound by the probe and cells not bound by the probe.

[0375] The probe may be specific and / or selective for the glycan. The probe may be a lectin, an antibody, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition or a cell. The probe may be monovalent.

[0376] The probe may be a lectin (either itself or attached to other structures).

[0377] The lectin may be an agglutinin. The agglutinin may be Ulex Europaeus Agglutinin I (UEA-I). UEA-I binds to glycans comprising a terminal fucose, such as an alpha- glycosidic linked fucose, for example an a(1^2) linked fucose linked to a galactose (e.g. Fuc al-2 Gal bl-4 GlcNAc).

[0378] The lectin may be Aleuria Aurantia Lectin (AAL). AAL binds to e.g. a Fuc a(1^6) GlcNAc, a Fuc a( 1 ^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc). AAL may bind to terminal alpha linked fucose glycans such as Fuc al -2 Gal [6S] b l -4 Glc.

[0379] The lectin may be Maackia Amurensis Lectin II (MAL-II). MAL-II binds to glycans comprising terminal sialic acids, such as an alpha glycosidic-linked sialic acid, for example an alpha-2, 3-linked sialic acid. MAL-II may bind to a terminal NeuAc a2-

[0380] 3 Gal b l -3 GalNAc residue.

[0381] The lectin may be Sambucus Nigra Lectin (SNA). SNA binds to glycans comprising terminal sialic acids, such as an alpha glycosidic-linked sialic acid, for example an alpha-2, 6-linked sialic acid. SNA may bind to a terminal NeuAc a2-6 Gal b l-

[0382] 4 GlcNAc residue.

[0383] The lectin may be Solanum Tuberosum Lectin (STL). STL binds to glycans comprising terminal N-acetylglucosamine, such as N-acetylglucosamine and / or oligomers of N- acetylglucosamine, and / or to glycan comprising internal N-acetyllactosamine. STL may bind to Chitin and / or (GlcNAc)3.

[0384] The lectin may be Narcissus Pseudonarcissus Lectin (NPL). NPL binds to glycans comprising a terminal alpha glycosidic-linked mannose, such as an alpha- 1, 6-linked mannose. NPL may bind to Man3GlcNAc2 and / or Man5GlcNAc2.

[0385] The lectin may be Galanthus Nivalis Lectin (GNA). GNA binds to glycans comprising a terminal alpha glycosidic-linked mannose, such as an alpha-1, 3-linked mannose. GNA may bind to Man3GlcNAc2.

[0386] The lectin may be Peanut Agglutinin (PNA). PNA binds to glycans comprising a terminal galactose, for example Fuc al -2 (Gal b l -3 GalNAc al -3) Gal b l -4 GlcNAc.

[0387] The lectin may be Styphnolobium japonicum (Japanese pagoda) lectin (SJA). SJA binds to glycans comprising a terminal galactose or terminal N-acetylgalactosamine, for example Fuc al -3 (Fuc al -2 (Gal al -3) Gal b l -4) GlcNAc or GalNAc b l -4 GlcNAc b l- 3 GalNAc b l -4 GlcNAc. The probe or lectin may be one or more selected from the group comprising MAL-II;

[0388] SNA; UEA-I; STL; NPL; AAL; GNA; PNA; SJA; and combinations thereof.

[0389] Thus, one or more lectins selected from the group consisting of MAL-II, SNA, NPL, GNA, AAL, UEA-I, PNA, SJA and STL may be used to detect, identify, enrich apoptotic or non-apoptotic cells and / or vesicles thereof, or to isolate apoptotic or non-apoptotic cells and / or vesicles thereof.

[0390] The terms N-acetylgalactosamine and GalNAc are used interchangeably herein.

[0391] The terms N-acetylglucosamine and GlcNAc are used interchangeably herein.

[0392] The term “detecting” referred to herein can mean determining the presence of one or more of the glycans referred to herein in a sample or subject. The detecting may comprise contacting the test sample with one or more probes that bind to the glycans referred to herein. Thus, detecting / detection may comprise contacting a mixture of non- apoptotic cells and apoptotic cells and / or vesicles thereof within a sample with one or more probes that bind to the glycans referred to herein. It is preferred that the probe is a “binding partner” (e.g. complementary binding partner) having affinity for said one or more glycan (e.g. an appropriate lectin, or appropriate lectins) and that the “detecting” step comprises detecting a level of glycan-binding partner complex (e.g. as a readout of the glycan residue level) formed following contact of the test sample with the one or more probe / binding partner. Lor example, the probe / binding partner may comprise a detectable agent (optionally as a conjugate) such as a fluorophore, a radioisotope or a dye (preferably a fluorophore). The level of such detectable agent can provide be used to measure the level of the complex.

[0393] The probe may be a probe referred to herein. Detection may be performed using any conventional assay or technique known in the art, including for example, but not limited to: flow cytometry; immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), an enzyme immunoassay (EIAs), radioimmunoassay (RIAs), Western Blots, immuno-precipitation or immunohistochemistry; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (ELIPR) assay; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS), or in vivo detection techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or radioactive labelling of lectins. Detection may be performed using flow cytometry or immunofluorescence. Detection may be performed using flow cytometry. Flow cytometry may be preferred because detection can be performed on the extracellular surface of live / intact cells or vesicles thereof.

[0394] When flow cytometry is used, a level of extracellular and / or extravesicular surface display of a glycan in the sample may be represented as percentage of cells (within a cell population) that are ‘positive’ for said extracellular and / or extravesicular glycan. In other words, a cell (and / or vesicle) that displays the glycan extracellularly (and / or extravesicularly) may be said to be “glycan positive”. For example, a “higher” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a higher percentage of “glycan positive” cells in the sample / test sample when compared with the reference sample. Similarly, an “lower” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a lower percentage of “glycan positive” cells in the sample / test sample when compared with the reference sample.

[0395] Additionally or alternatively, and preferably where the probe is conjugated to a detectable agent (e.g. fluorophore), a level of extracellular and / or extravesicular surface display of a glycan in the sample may be represented as a “mean fluorescence intensity” (MFI) value. This term is well known to the skilled person in the art, and such MFI value is understood to be indicative of antigen abundance (e.g. for the presence circumstances, glycan abundance). For example, a “higher” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a higher “MFI” value in the sample / test sample when compared with the reference sample. Similarly, a “lower” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a lower “MFI” value in the sample / test sample when compared with the reference sample.

[0396] The term “mean” here (in the context of MFI) can be used interchangeably with the terms “median”, “arithmetic mean”, “geometric mean” and “mode”. The “sample ” or “test sample ” may be a biological sample or a bodily sample of a subject (a sample taken from a subject, such as human or any other organism). The sample may be a bodily sample. Thus, the invention may be carried out in vitro. The sample may comprise blood (e.g., umbilical cord blood), plasma, serum, bone marrow, amniotic fluid, adipose or other tissues, or combinations thereof. Thus, the sample may be recovered from blood, bone marrow, amniotic fluid, dental pulp of molar cells, an umbilical cord blood, adipose tissue, or any other tissue or bodily fluid or combinations thereof. Alternatively, the sample may be an ex vivo sample or an in vitro sample. Therefore, the cells may be in a tissue or organ sample (for ex vivo based tests) or the cells may be grown in culture (an in vitro sample) or in the blood, tissue or body (an in vivo sample). The sample may or may not have undergone in vitro expansion. The sample may be “a primary cell culture ”. The methods disclosed herein may performed in vivo (e.g., via blood plasma apheresis system or PET imaging). The sample or test sample may comprise a mixture of non-apoptotic cells and apoptotic cells and / or vesicles thereof. All the cells may be live cells. In embodiments wherein the invention is performed in vivo, the test sample may be the subject per se. The sample may comprise a mixture of cells. The sample may comprise a mixture of apoptotic cells and / or vesicles thereof, which display a glycan on their external surface, together with non-apoptotic cells and / or vesicles thereof.

[0397] The invention may be used to detect, enrich or remove apoptotic cells and / or vesicles thereof from a sample. Vesicles of apoptotic cells may be detected by detecting extravesicular surface display of one or more of the glycans referred to herein. The “sample ” may be a biological sample or a bodily sample of a subject (a sample taken from a subject, such as human or any other organism). Thus, the invention may be carried out in vitro. Methods of the invention are preferably in vitro methods. Thus, the sample may be an in vitro sample. Therefore, the cells may be grown in culture. The sample may or may not have undergone in vitro expansion. The sample may be “a primary cell culture ”. The sample may comprise a mixture of cells. The sample may comprise a mixture of apoptotic cells and / or vesicles thereof (including cells in which caspase 3 has been activated), which display a glycan on their external surface, together with non-apoptotic cells and / or vesicles thereof.

[0398] Vesicles referred to herein may be a structure that is located inside or outside a cell or fused with a cell and comprises a lipid bilayer that encloses a liquid, such as a cytoplasm, and / or bioactive molecules, and / or RNA. Vesicles referred to herein may be one or more structures selected from the group consisting of lysosomes, extra-cellular vesicles, microvesicles, nano-vesicles, exosomes, apoptotic bodies, oncosomes, enucleated cells, artificial vesicles or similar structures.

[0399] Preferably, a “vesicle” comprises an outer membrane derived from the outer membrane of an apoptotic cell from which the vesicle is released. This allows advantage to be taken of the observation that outer cell-membrane glycans provide a readout of apoptosis.

[0400] The reference sample or negative control may be a sample that does not comprise or contain apoptotic cells and / or vesicles thereof (e.g., non-apoptotic cells and / or vesicles thereof). The reference sample may be a sample of cells and / or vesicles thereof. The reference sample (or negative control) may be a sample that comprises cells and / or vesicles thereof that do not display any of the glycan signature referred to herein. The reference sample may be a sample of (control) cells and / or vesicles thereof. The reference sample may comprise viable cells. The reference sample may be from the same or a different subject as the test sample. The test sample may comprise the same type of cells as the reference sample. The test sample may be matched with a reference sample of the same age. There may be about at 1 to 5 -year difference between the reference sample and the test sample.

[0401] The reference level may be a reference level of extracellular and / or extravesicular surface display of the glycan in a reference sample.

[0402] The “subject” referred to herein may be an animal or a plant. The animal may be a vertebrate, a mammal, or a domestic animal. The subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc. The subject may be a human.

[0403] Furthermore, “apoptotic cells” (and / or vesicles thereof), for example as described in methods of the invention, may be animal or a plant cells. The “apoptotic cells” (and / or vesicles thereof) may be vertebrate, mammal, or domestic animal cells. The “apoptotic cells” (and / or vesicles thereof) may be from any animal of veterinary interest, for instance, a cat, dog, horse etc. Preferably, the “apoptotic cells” (and / or vesicles thereof) are human cells. All of these standard taxonomic definitions may likewise apply to a “non-apoptotic” cell described herein.

[0404] It will be appreciated that the term “treatment” and “treating” as used herein means the management and care of a subject for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, including alleviating symptoms or complications, delaying the progression of the disease, disorder or condition, alleviating or relieving the symptoms and complications, and / or to cure or eliminating the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a subject for the purpose of combating the disease, condition, or disorder and includes the administration of the ligand to prevent the onset of the symptoms or complications.

[0405] The invention may be used in vivo, in vitro or ex vivo. Thus, the method disclosed herein may be performed in in vivo, in vitro or ex vivo,' preferably in vitro or ex vivo. The kit may be used in vivo, in vitro or ex vivo,' preferably in vitro or ex vivo.

[0406] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0407] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures.

[0408] Figure 1 - Early fibroblast lectin changes after staurosporine treatment in Annexin V negative cells

[0409] Fibroblasts were subjected to staurosporine treatment over 6 hours. DMSO vehicle control and treatment time-points at 1 hour, 3 -hours and 6-hours were analysed for lectin binding by AAL, GNA, MAL-II, NPL, SNA and STL. Normalised lectin gMFI at each time-point is expressed as a percentage relative to that of the DMSO vehicle control. 100% dotted line indicate no change (A). Annexin V percentage positive value is indicated for the whole population (B). Ratios of selected lectin bindings are expressed as a percentage of that for vehicle control (C). Annexin V negative subpopulations are shown in (A and C).

[0410] Figure 2 - Early MSC lectin changes after staurosporine treatment in Annexin V negative cells

[0411] Bone marrow MSC (BMMSC) were subjected to staurosporine treatment over 7 hours. DMSO vehicle control and treatment time-points at 1 hour, 3-hours and 7-hours were analysed for lectin binding by AAL, GNA, MAL-II, NPL, SNA and STL. Normalised lectin gMFI at each time-point is expressed as a percentage relative to that of the DMSO vehicle control. 100% dotted line indicate no change (A). Annexin V percentage positive value is indicated for the whole population (B). Ratios of selected lectin bindings are expressed as a percentage of that for vehicle control (C). SJA and PNA binding were studied separately over 7 hours. Lectin binding at each time-point is expressed as gMFI for SJA (D) and PNA (E). Annexin V percentage positive value is indicated for the whole population (F). Annexin V negative subpopulations are shown in (A, C, D and E).

[0412] Figure 3 - Early MSC lectin changes after treatment with anti-FAS antibody in Annexin V negative cells

[0413] BMMSC were subjected to anti-FAS antibody for 6 hours. DMSO vehicle control and treatment groups were analysed for lectin binding by AAL, GNA, MAL-II, NPL, SNA and STL. Normalised lectin gMFI at each time-point is expressed as a percentage relative to that of the DMSO vehicle control. 100% dotted line indicate no change (A). Annexin V percentage positive value is indicated for the whole population (B). Ratios of selected lectins are expressed as a percentage of that for vehicle control (C). Annexin V negative subpopulations are shown in (A and C).

[0414] Figure 4 - Early HUVEC (Human umbilical vein endothelial cells) lectin changes after staurosporine treatment in Annexin V negative cells

[0415] Human umbilical vein endothelial cells (HUVEC) were subjected to staurosporine treatment over 7 hours. DMSO vehicle control and treatment time-points at 1 hour, 3- hours and 7-hours were analysed for lectin binding by AAL, GNA, MAL-II, NPL, SNA, STL and UEA-I. Normalised lectin gMFI at each time-point is expressed as a percentage relative to that of the DMSO vehicle control. 100% dotted line indicate no change (A). Annexin V percentage positive value is indicated for the whole population (B). Ratios of selected lectins are expressed as a percentage of that for vehicle control

[0416] (C). Annexin V negative subpopulations are shown in (A and C).

[0417] Figure 5 - Gating Strategy

[0418] Cells were gated according to figure (a). Single cells are defined by FSC-A and FSC- H parameters (b). Alive cells are defined as 7AAD negative (c). Two subpopulations of Annexin intensity are defined as Annexin negative and Annexin high (d).

[0419] All samples shown, unless otherwise specified, are from the Annexin negative gate.

[0420] Materials and Methods

[0421] Cell culture

[0422] Bone marrow derived mesenchymal stem cells (BMMSC; Biorooster), were grown in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% of Fetal Bovine Serum (FBS; 12,483,040; Thermo Fisher Scientific). Human fibroblasts (StemNovate) were cultured using a ready to use fibroblast growth medium (C-23025; Promocell). Human umbilical vein endothelial cells (HUVEC; PromoCell) were cultured using ready to use endothelial cell growth medium (C-22010; Promocell). All different cell types were incubated in standard condition (37°C with 5% CO2).

[0423] Surface marker expression - cell identity

[0424] Bone marrow MSC were tested for the expression of the following markers: CD45, CD73, CD90, CD105 and found to be positive for CD73, CD90 and CD105 and negative for CD45 (Table 1), as is common for MSC. For the HUVEC characterization we used the following markers: CD31, CD45 and CD 146 and found to be positive for CD31 and CD 146 and negative for CD45 (Table 1), as is common for endothelial cells. Finally, the human fibroblasts were tested positive for the CD90 and negative for CD45 surface markers (Table 1), as is common for fibroblasts.

[0425] The HUVEC and the fibroblast populations from 3 different donors were pooled.

[0426] BMMSC (n=l individual donor).

[0427] Extrinsic and intrinsic apoptosis induction

[0428] Extrinsic apoptosis was induced on MSC by anti-FAS monoclonal antibody (aFAS; CHI 1; Merck) treatment followed by 6 hours incubation of cells for sufficient apoptosis induction. Untreated (control) MSC were also included.

[0429] Intrinsic apoptosis was induced on MSC, fibroblasts and HUVECs by treatment of with IpM Staurosporine (SM97-1; Cambridge Bioscience) for either 1, 3 or 6 hours (fibroblasts) or 1, 3 or 7 hours (MSC and HUVECs). Vehicle control samples were treated with equal concentration of DMSO (solvent). Upon apoptosis induction, cells were incubated in a humidified incubator (37°C with 5% CO2).

[0430] Lectin staining / AnnV staining and flow cytometry analysis

[0431] To assay cells, after washing adhering cells, adherent cells were detached using known treatment protocols (for detachment) and briefly incubated at culture temperature prior to lectin staining. Lectins were used to stain glycans on the surface of different cell populations followed by incubation of cells with PE conjugated secondary antibody, prior to flow cytometry analysis. This was conducted following standard parameters with normal configurations suited to flow cytometry. The lectins were titrated to prevent agglutination (as per standard practice). Flow cytometric analysis was normalised by subtracting secondary only control staining. Briefly, BMMSC, HUVECs or fibroblasts were detached from flasks and cells washed twice in PBS (300g, 5min) prior determination of cell numbers. Cells were then incubated in blocking buffer for lOmin and washed twice (300g, 5min). Subsequently, cells were incubated in blocking buffer containing viability dye (554061 ; BD Biosciences) for 15min and washed (300g, 5min). In the meantime, lectins were diluted in blocking buffer at the appropriate concentrations for the best signal between conditions. Following staining with the viability dye, about 5xl05cells per condition were washed twice in blocking buffer (300g, 5min) and incubated in lectin containing buffers for 30min at 4°C. Cells were then washed once in blocking buffer and incubated with PE-conjugated secondary antibody, as well as AnnV-FITC antibody (556419; BD) following manufacturer’s recommendations, for 30min at 4°C. Finally, cells were washed (300g, 5min) and analysed with the flow cytometer.

[0432] Statistical analysis

[0433] Statistical analyses were performed using Prism 9 (Graph Pad) using Student’s t-test or one-way Anova when appropriate. Significance was reached at p<0.05. Additional significances are shown as * p < 0.05 * * p < 0.01 * * p < 0.001 * * * * p < 0.0001.

[0434] Examples

[0435] Initiation of apoptosis triggered through the extrinsic pathway via anti-Fas antibody and the intrinsic pathway via staurosporine. Following initiation of apoptosis specific lectin- binding profiles were established on an Annexin V negative populations of primary cells (i.e., bone marrow derived MSC, fibroblasts and HUVEC).

[0436] One of the consistent lectin binding profiles observed following initiation of apoptosis was 1) rise in SNA binding, this was in the first few hours post apoptosis initiation and prior to the induction of AnnV, indicating increased exposure of a-2,6 sialic acids; 2) decline of GNA and NPL binding showing a fall in exposure of terminal mannose glycans; 3) decline in AAL binding indicating a fall in alpha fucose terminating glycans; and 4) a decline in STL binding indicating a fall in GlcNAc terminating glycans.

[0437] Changes to MAL-II binding, indicating exposure of a-2,3 sialic acids, was also seen.

[0438] Example 1- Intrinsic apoptosis induces early-stage cell surface glycan fluctuations in fibroblasts before phosphatidyl serine exposure

[0439] Intrinsic apoptosis induction on fibroblast cells by staurosporine was analysed for changes to plant lectin binding within the first 6 hours (Figure 1), where Annexin V binding is not fully activated (Figure IB). SNA lectin, exhibits a 20% spike in binding to fibroblasts at 1 hour timepoint of staurosporine treatment within the Annexin negative cell population (Figure 1A). From the first hour to hour 6, SNA binding decreases to beneath the baseline. (Figure 1A). By clear contrast, AAL, STL, MAL-II, NPL and GNA, all show time dependent decline in binding over the first six hours post staurosporine treatment (Figure 1A). Importantly this is a time period where Annexin V binding has not been fully stimulated by staurosporine (Figure IB). An apparent phosphatidyl independent signature of glycosylation fluctuation is therefore reported in the early, pre-commitment phase of apoptosis. Ratios of selected lectins further demonstrate great sensitivity in the pre-commitment phase of apoptosis (Figure 1C).

[0440] Example 2 - Bone marrow derived mesenchymal stem cells (BMMSC) mirror fibroblasts in exhibiting early plant lectin binding divergence during intrinsic and extrinsic apoptosis

[0441] In comparison to fibroblasts, BMMSC demonstrate similar overall trends in lectin binding divergence between SNA and other lectins, but with subtle differences. SNA still exhibit the same binding spike at hour one of staurosporine treatment, but quite dramatically declines to below 50% of initial levels over the full course of 7-hours (Figure 2A). Binding of SJA is absent in the Vehicle control and after 1 hour of staurosporine stimulation (Figure 2D). SJA binding rises dramatically, peaking at 3 hours and this high binding is sustained up to 7 hours (Figure 2D). PNA binding rises by the 1sthour but dramatically reduces at 3 hours and makes a recovery at 7 hours. GNA, MAL-II, NPL, STL and AAL, all consistently show decrease in binding over the 7-hours of staurosporine treatment in both Annexin V negative subpopulations (Figure 2A). Ratios of selected lectins further demonstrate great sensitivity in the precommitment phase of apoptosis (Figure 2C). Extrinsic apoptosis was induced by anti-Fas antibody and lectin staining reveal a similar profile in glycan changes at the pre-commitment phase of early apoptosis: rise in SNA and decline in other lectins, notably NPL and GNA (Figure 3A). 6-hour stimulation was selected to test lectin binding prior to full phosphatidyl serine exposure, typically found at 24-hours (Figure 3B). STL binding however, does not fluctuate (Figure 3A) in the Annexin V negative population. Ratios of selected lectins further demonstrate great sensitivity in the pre-commitment phase of apoptosis (Figure 3C).

[0442] Example 3 - HUVEC exhibiting glycan changes during early intrinsic apoptosis

[0443] In comparison to fibroblasts and BMMSC, HUVEC profiles show largely the same trend where most lectin binding is declining in the pre-Annexin V induction phase in the Annexin Negative population (Figure 4A). No significant increase in Annexin V binding was observed over 7-hour of staurosporine treatment, thus this timeframe represents the pre-apoptotic commitment phase (Figure 4B). One difference to other cell-types is that AAL binding is spiking at all three time points and UEA-I, STL, SNA and MAL-II binding spike only at the first hour. NPL and GNA binding is consistently declining with time and at 7-hours MAL-II binding follows the same trend (Figure 4A). These results show that similar patterns of glycosylation changes may be shared between various cell types while specific lectin behaviour could present cell-type specific signatures. Ratios of selected lectins further demonstrate great sensitivity in the precommitment phase of apoptosis (Figure 4C).

[0444] Surprisingly, UEA-I does not bind fibroblasts or BMMSC, but binds HUVEC. A mild increase is observed in HUVEC UEA-I binding in the Annexin negative population after one hour of staurosporine stimulation. This result demonstrates that changes in HUVEC specific lectin, UEA-I binding, is a biomarker for pre-apoptotic endothelial cells.

Claims

Claims1. A method of detecting apoptotic cells and / or vesicles thereof in a sample, the method comprising: providing a sample, determining the level of extracellular and / or extravesicular surface display of a glycan in the sample, wherein a difference in the level of extracellular and / or extravesicular surface display of the glycan detected in the sample compared to a reference level is indicative that the sample comprises apoptotic cells and / or vesicles thereof.

2. A method of detecting the presence or absence of apoptotic cells and / or vesicles thereof in a sample, the method comprising: providing a sample, determining a level of extracellular and / or extravesicular surface display of a glycan in the sample, comparing the level of display of the glycan in the sample with a reference value, and detecting the presence of apoptotic cells and / or vesicles thereof, or detecting the absence of apoptotic cells and / or vesicles in the sample when based on the comparison.

3. The method of claim 1 or claim 2, wherein the method comprises detecting the level of extracellular and / or extravesicular display of a glycan about 1 hour or earlier to about 4 hours, about 1 hour or earlier to about 3.5 hours, about 1 hour or earlier to about 3 hours, or 1 hour or earlier to about 2 hours after the initiation of apoptosis.

4. The method according to any one of claims 1-3, wherein the glycan comprises one or more residues selected from the group consisting of:(i) terminal sialic acid residue(s),(ii) terminal galactose residue(s),(iii) terminal mannose residue(s),(iv) terminal N-acetylgalactosamine (GalNAc) residue(s),(v) terminal fucose residue(s),(vi) terminal N-acetylglucosamine (GlcNAc) residue(s), and(vii) internal N-acetyllactosamine residue(s); preferablywherein detecting comprises contacting the test sample with one or more probes that bind the glycan.

5. The method according to any one of the preceding claims, wherein detecting is performed using any method, such as flow cytometry; immunoassays, such as enzyme- linked immunosorbent assays (ELISAs), an enzyme immunoassay (EIAs), radioimmunoassay (RIAs), Western Blots, immuno-precipitation or immunohistochemistry; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (FLIPR) assay; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS), or in vivo detection techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or radioactive labelling of lectins.

6. The method according to any one of the preceding claims, wherein detecting is performed by flow cytometry.

7. The method according to any one of the preceding claims, wherein the difference in the level of display of the glycan in the sample relative to the level of display of the glycan in the reference sample is a higher level of display or a lower level of display.

8. The method according to any one of claims 2-7, wherein:(i) the ‘presence’ of apoptotic cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘the same’ or Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising terminal mannose is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

9. The method according to claim 8, wherein said terminal mannose is an alpha-1.3-linked mannose or an alpha- 1,6-linked mannose; preferably wherein said terminal mannose is Man3GlcNAc2 or Man5GlcNAc2.

10. The method according to any one of claims 2-9, wherein:(i) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N- acetylglucosamine is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2.3-linked sialic acid, terminal fucose, terminal mannose and terminal N- acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N- acetylglucosamine is ‘the same’ or Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N- acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

11. The method according to any one of claims 2-10, wherein:(i) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6- linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

12. The method according to any one of claims 2-11, wherein:(i) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

13. The method according to any one of claims 2-12, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the groupconsisting of a terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose and terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

14. The method according to any one of claims 2-13, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha- 2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

15. The method according to any one of claims 2-14, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the levelof extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha- 2, 6-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

16. The method according to any one of claims 2-15, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 3 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 3 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 3 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N- acetylgalactosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 3 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminalN-acetylgalactosamine is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

17. The method according to any one of claims 2-16, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours postinitiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours postinitiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours postinitiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours postinitiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

18. The method according to any one of claims 2-17, wherein:(i) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic stem cells, such as mesenchymal stem cells, (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

19. The method according to any one of claims 2-18, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal fucose residue is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal fucose residue is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal fucose residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycancomprising a terminal fucose residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof). For example, where the apoptotic cells (and / or vesicles thereof) are defined as apoptotic cells at up to seven hours post-initiation.

20. The method according to any one of claims 2-19, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 3- linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

21. The method according to any one of claims 2-20, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / orextravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 3-linked sialic acid is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 3-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

22. The method according to any one of claims 2-21, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal alpha-2, 6- linked sialic acid is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal alpha-2, 6-linked sialic acid is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

23. The method according to any one of claims 2-22, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N- acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N- acetylglucosamine is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof); or(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N- acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N- acetylglucosamine is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

24. The method according to any one of claims 2-23, wherein:(i) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is Tower’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(ii) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does not comprise an apoptotic cell (or vesicle thereof);(iii) the ‘presence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal N-acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof); or(iv) the ‘absence’ of apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise an apoptotic cell (or vesicle thereof).

25. The method according to any one of claims 10-24, wherein said terminal alpha- 2,3-linked sialic acid is NeuAc a2-3 Gal b 1-3 GalNAc.

26. The method according to any one of claims 10-25, wherein said terminal fucose is an alpha-glycosidic linked fucose; such as a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc); or an a( 1— >2) linked fucose linked to a galactose.

27. The method according to any one of claims 10-26, wherein said terminal mannose is an alpha-1, 3-linked mannose or an alpha- 1,6-linked mannose; preferably wherein said terminal mannose is Man3GlcNAc2 or Man5GlcNAc2.

28. The method according to any one of claims 10-27, wherein said terminal N- acetylglucosamine is P(l,4)-linked GlcNAc, or oligomer thereof such as Chitin and / or (G1CNAC)329. The method according to any one of claims 11-28, wherein said alpha-2, 6-linked sialic acid is a NeuAc a2-6 Gal bl-4 GlcNAc residue.

30. The method according to any one of claims 16-29, wherein said terminal N- acetylgalactosamine is a P-N-Acetylgalactosamine; preferably Fuc al-3 (Fuc al-2 (Gal al-3) Gal bl-4) GlcNAc.

31. The method according to any one of claims 17-30, wherein said terminal galactose is a galactosyl (P-1,3) 7V-acetylgalactosamine; preferably Fuc al-2 (Gal bl-3 GalNAc al -3) Gal bl -4 GlcNAc.

32. The method according to any one of the preceding claims, further comprising the step of recording on a suitable data carrier, data obtained in the step of determining the level of extracellular and / or extravesicular surface display of a glycan in the sample.

33. A kit for detecting apoptotic cells and / or vesicles thereof in a sample of cells, the kit comprising: a probe for a glycan; wherein the glycan comprises one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues; wherein the probe of the kit is used to determine, in a test sample, the level of display of one or more of the glycans, wherein a difference in the level of display of one or more of the glycans in the test sample relative to a level of display of one or more of the glycans in a reference sample, indicates the presence of apoptotic cells and / or vesicles thereof in the test sample, optionally this can be used in the diagnosis of an apoptotic-related disease.

34. A kit according to claim 33, wherein the probe is conjugated to an imaging label.

35. A kit according to claim 33 or claim 34, wherein the kit comprises a further agent for detecting a further biomarker of apoptosis.

36. A method of enriching apoptotic or non-apoptotic cells in a sample, the method comprising:contacting the sample, which comprises a mixture of non-apoptotic cells and apoptotic cells, with a probe for a glycan; and enriching the non-apoptotic or the apoptotic cells using the probe wherein the probe is specific for residues on the glycan selected from the group consisting of(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues.

37. A kit for enriching for non-apoptotic cells and / or vesicles thereof, or apoptotic cells and / or vesicles thereof, in a sample, the kit comprising: a separation means for separating non-apoptotic cells and / or vesicles thereof from apoptotic cells and / or vesicles thereof; or a separation means for separating apoptotic cells and / or vesicles thereof from non-apoptotic cells and / or vesicles thereof; and a probe for a glycan; wherein the probe is directed to glycans comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues; wherein the probe binds to apoptotic cells and / or vesicles thereof that extracellularly and / or extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (non-apoptotic) cells from bound (apoptotic) cells and / or vesicles thereof, thereby enriching the non- apoptotic cells and / or vesicles thereof within the test sample, orwherein the probe binds to non- apoptotic cells and / or vesicles thereof that extracellularly and / or extravesicular display on their surface a glycan comprising terminal residues selected from the group consisting of (i) to (vii) and the separation means uses the probe to separate unbound (apoptotic) cells and / or vesicles thereof from bound (non-apoptotic) cells and / or vesicles thereof, thereby enriching the apoptotic cells and / or vesicles thereof within the test sample.

38. The method according to claim 36, or the kit according to claim 37, wherein: the probe binds to non-apoptotic cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal fucose, such as a terminal alpha- 1,6-linked mannose and / or a terminal alpha-1, 3-linked mannose, optionally wherein the probe is selected from NPL and GNA; the probe binds to non-apoptotic connective tissue cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3- linked sialic acid, terminal fucose, terminal mannose such as a terminal alpha- 1,6-linked mannose and / or a terminal alpha-1, 3-linked mannose and / or terminal N-acetylglucosamine; optionally wherein the probe is selected from MAL-II, AAL, GNA, NPL and STL; the probe binds to non-apoptotic mesenchymal stem cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3-linked sialic acid, terminal fucose, terminal mannose such as a terminal alpha- 1,6-linked mannose and / or a terminal alpha-1, 3-linked mannose and / or terminal N-acetylglucosamine, optionally wherein the probe is selected from MAL-II, AAL, GNA, NPL and STL; the probe binds to apoptotic endothelial cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal fucose, optionally wherein the probe is selected from AAL and UEA-1 ; the probe preferentially binds non-apoptotic connective tissue cells (and / or vesicles thereof) over apoptotic connective tissue cells (and / or vesicles thereof) that are at > 1 hour post-initiation by binding extracellular (and / or extravesicular) displayed terminal alpha-2, 6-linked sialic acid, optionally wherein the probe is SNA; the probe preferentially binds non-apoptotic mesenchymal stem cells (and / or vesicles thereof) over apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at > 1 hour to < 7 hours post-initiation by binding extracellular(and / or extravesicular) displayed terminal galactose, optionally wherein the probe is PNA; the probe preferentially binds non-apoptotic endothelial cells (and / or vesicles thereof) over apoptotic endothelial cells (and / or vesicles thereof) that are at > 7 hours post-initiation by binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 3-linked sialic acid and / or terminal N-acetylglucosamine, optionally wherein the probe is selected from MAL-II and STL; the probe preferentially binds apoptotic connective tissue cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation by binding extracellular (and / or extravesicular) displayed terminal alpha-2, 6-linked sialic acid, optionally wherein the probe is SNA; the probe preferentially binds apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at > 3 hours post-initiation by binding extracellular (and / or extravesicular) displayed terminal N-acetylgalactosamine, optionally wherein the probe is SJA; the probe preferentially binds apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours post-initiation by binding extracellular (and / or extravesicular) displayed terminal galactose, optionally wherein the probe is PNA; the probe preferentially binds apoptotic mesenchymal stem cells (and / or vesicles thereof) that are at up to (and including) 1 hour or > 7 hours post-initiation by binding extracellular (and / or extravesicular) displayed terminal galactose, optionally wherein the probe is PNA; the probe preferentially binds apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation by binding extracellular (and / or extravesicular) displayed terminal alpha-2, 3-linked sialic acid, optionally wherein the probe is MAL-II; and / or the probe preferentially binds apoptotic endothelial cells (and / or vesicles thereof) that are at up to (and including) 1 hour post-initiation by binding extracellular (and / or extravesicular) displayed: terminal alpha-2, 6-linked sialic acid and / or terminal N-acetylglucosamine, optionally wherein the probe is selected from SNA and STL.

39. A method of diagnosing an apoptotic-related disease or disorder in a subject, the method comprising:detecting, in the sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, wherein detection, in the sample, of a glycan comprising terminal residues selected from the group consisting of (i) to (vii) is indicative that the subject has an apoptotic-related disease or disorder.

40. A method of treating or preventing an apoptotic disease or disorder in a subject, the method comprising: enriching non-apoptotic cells in a sample comprising a mixture of apoptotic and non-apoptotic cells using a method according to claim 35 or a kit according to claim 38; and administering the enriched sample of non-apoptotic cells to the subject to treat the disease or disorder.

41. The method according to claim 39 or 40, wherein the disease or disorder is a cardiovascular disease such as myocardial infarction, a neurodegenerative disease such as Alzheimer’s, cancer, autoimmunity, liver disease, type 1 diabetes, retinal degeneration, HIV / AIDS and others.

42. An apoptotic cell probe-agent conjugate, the conjugate comprising: a probe for targeting and / or binding to a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, conjugated to an agent.

43. The method of claim 41, wherein a conjugate is used to visualise a apoptotic cell, or tag it for detection, elimination or enrichment, preferably the probe is conjugated to an agent, such as an imaging agent, to provide a probe-agent conjugate.

44. A conjugate according to claim 42, wherein the agent is an imaging agent.

45. A method of visualizing apoptotic cells and / or vesicles thereof in a sample, the method comprising: administering a probe-agent conjugate according to claim 44 to the sample, wherein the agent comprises an imaging agent; and detecting the imagining agent in the subject in order to visualise apoptotic cells and / or vesicles thereof in the sample.

46. The conjugate of claim 44 or the method of claim 45, wherein the imaging agent is a fluorophore, a radioisotope, or a dye.

47. The method of claim 45 or claim 46, wherein detecting the imaging agent is performed using a whole body imagine system, such as an MRI scan.

48. The method, kit or a conjugate of any preceding claim, wherein the probe is a lectin.

49. The method, kit or a conjugate of claim 48, wherein the lectin is one or more selected from the group consisting of MAL-II; SNA; UEA-I; STL; NPL; AAL; GNA; PNA; SJA; any other lectin that recognizes the same glycan as one or more of MAL-II, SNA, UEA-I, STL, NPL, AAL, GNA, PNA, and SJA; and combinations thereof.

50. The method, kit, conjugate, probe, use or method of any preceding claim wherein the sialic acid residues are alpha linked sialic acid residues.

51. The method, kit, conjugate, probe, use or method of claim 50, wherein the alpha linked sialic acid residues are alpha-2, 3-linked sialic acids,52. The method, kit, conjugate, probe, use or method of claim 51, wherein the alpha- 2,3-linked sialic acids are NeuAc a2-3 Gal b 1-3 GalNAc residues.

53. The method, kit, conjugate, probe, use or method of claim 50, wherein the alpha linked sialic acid residues are alpha-2, 6-linked sialic acids.

54. The method, kit, conjugate, probe, use or method of claim 53, wherein the alpha-2, 6-linked sialic acid residues are NeuAc a2-6 Gal bl -4 GlcNAc.

55. The method, kit, conjugate, probe, use or method of any preceding claim wherein the galactose residues are Fuc al -2 (Gal bl -3 GalNAc al -3) Gal bl -4 GlcNAc or Fuc al-3 (Fuc al-2 (Gal al-3) Gal b 1-4) GlcNAc.

56. The method, kit, conjugate, probe, use or method of any preceding claim, wherein the mannose residues are alpha linked mannose residues, such as alpha- 1,6- linked mannose residue and / or an alpha-1, 3-linked mannose residue which can optionally be Man3GlcNAc2 and / or Man5GlcNAc2.

57. The method, kit, conjugate, probe, use or method of any preceding claim wherein the N-acetylgalactosamine residues are GalNAc bl -4 GlcNAc bl -3 GalNAc bl -4 GlcNAc.

58. The method, kit, conjugate, probe, use or method of any preceding claim wherein the fucose residues are alpha-linked fucose residues, such as a(1^2) linked fucose linked to a galactose; or a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1^6) GlcNAc).

59. The method, kit, conjugate, probe, use or method of any preceding claim wherein the N-acetylglucosamine residues are Chitin or (GlcNAcjs.

60. The method, kit, conjugate, probe, use or method of any preceding claim wherein the internal N-acetyllactosamine residues are poly-LacNAc.

61. Use of an extracellular and / or extravesicular surface displayed glycan as an apoptotic cell biomarker.

62. Use of a lectin to detect apoptotic cells and / or vesicles thereof or enrich apoptotic cells or non-apoptotic cells.

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