Cell state detection

Abstract

The invention provides a method of characterising the state of a particle comprising a cell membrane, the method comprising: determining the presence of a glycan first marker on a surface of the particle; determining the presence of a second marker associated with the particle; and characterising the state of the particle on the basis of these determinations.

Description

[0001] CELL STATE DETECTION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods for characterising the state of particles, or populations of particles, comprising a cell membrane. The invention also relates to kits useful in such methods of characterising the state of such particles.

[0004] SUMMARY OF THE INVENTION

[0005] In a first aspect, the invention provides a method of characterising the state of a particle comprising a cell membrane, the method comprising:

[0006] • determining the presence of a glycan first marker on a surface of the particle;

[0007] • determining the presence of a second marker associated with the particle; and characterising the state of the particle on the basis of these determinations.

[0008] In a second aspect, the invention provides a method of characterising the state of a population of particles comprising a cell membrane, the method comprising:

[0009] • determining the presence of a glycan first marker on a surface of particles within a population;

[0010] • determining the presence of a second marker associated with particles within a population; and

[0011] characterising the state of the population of particles on the basis of these determinations.

[0012] Suitably, the presence of the first and second marker is determined in respect of particles within the same population.

[0013] In a third aspect, the invention provides a method of characterising the state of a first and / or second particle comprising a cell membrane, the method comprising:

[0014] • determining the presence of a glycan first marker on a surface of a first particle; • determining the presence of a second marker associated with the first particle; and • determining the presence of the glycan first marker on a surface of a second particle;

[0015] • determining the presence of the second marker associated with the second particle;

[0016] and

[0017] • comparing the results achieved in respect of the first and second markers in the first and second particles, andcharacterising the state of the first and / or second particle on the basis of these determinations.

[0018] In a fourth aspect, the invention provides a method of characterising the state of a first and / or second population of particles comprising a cell membrane, the method comprising:

[0019] • determining the presence of a glycan first marker on a surface of particles within a first population;

[0020] • determining the presence of a second marker associated with particles within the first population; and

[0021] • determining the presence of the glycan first marker on a surface of particles associated with a second population;

[0022] • determining the presence of the second marker associated with particles of the second population; and

[0023] • comparing the results achieved in respect of the first and second markers in the first and second populations of particles, and

[0024] characterising the state of the first and / or second population of particles on the basis of these determinations.

[0025] Suitably the presence of the first glycan marker is determined on the external surface of a particle, or of particles in a population.

[0026] The skilled person would readily appreciate that a characterisation step can benefit from comparison of the determinations outlined in the aspects above with a reference standard, the latter comprising corresponding determinations for a control particle (or a population of particles) of known state. The state of a particle (or particles) may be characterised accordingly as being the same state as the control particle(s), when the determinations for the first and second markers are equivalent to the reference standard. Additionally or alternatively, a particle (or particles) may be characterised as ‘not’ being in the same state as the control particle(s), when the determinations for the first and second markers are not equivalent to the reference standard. Naturally, a plurality of reference standards (for a plurality of different states) may be employed, particularly in methods that involve a population of particles, that may comprise a heterogenous population of states.

[0027] Such determination for a reference standard may be conducted as an active step of a method described herein. That being said, such determinations for the reference standard are preferably provided (e.g. from an external source) based on previously conducted determinations, e.g. carried out under equivalent conditions to a method of the invention.Methods of the invention may involve assaying a particle or particles (e.g. in a test sample) that are putatively of the same state as a control particle of known state (represented by the reference standard). For example, a particle (or particles) may be putatively apoptotic, with a control particle being of a known apoptotic state (e.g. pre-commitment state, commitment state or otherwise) to allow for characterisation of the particular apoptotic state that the (assayed) particle or particles belong to.

[0028] That being said, methods of the invention may be totally agnostic, and may involve assaying a particle or particles whose state is unknown (e.g. not associated with a putative state characterisation), and a reference standard (for a particle that ‘does’ happen to be of known state) can thus be used to characterise the state of an assayed particle e.g. in an agnostic manner.

[0029] As will be outlined in more detail below, such determinations may indicate a level for the first and second markers (indeed, it is preferred the reference to determining the presence of a marker herein includes determining a level for said marker). Thus, a reference standard may indicate a level for each of said markers that is known to be associated with a particular (known) state.

[0030] As discussed below, such levels (first and second) can be provided as 2D data points on a plot, thus the reference standard may indicate the corresponding 2D data point for a control particle of known. Equivalent positions (on the 2D plots) for the 2D data point of an assayed particle and of a control particle may thus allow for characterisation of the assayed particle accordingly, i.e. as being the same state as the control particle; or non-equivalent positions may characterise the assayed particle as not being of the same state as the control particle. The skilled person understands how to determine equivalence in this context, and appreciates that equivalent positions do not need to share precisely the same first and second axis (e.g. X and Y axis) values, so long as they share sufficiently significant statistical nexus.

[0031] As discussed below, for embodiments involving a plurality of particles (each yielding a 2D data point), a contour plot may be provided. Thus, a reference standard may provide a corresponding control contour plot, having a contour for a population of control particles of known state (or a plurality of contours for populations of different (but known) states). Equivalent positions (on the contour plot) for a contour for an assayed population of particles and a contour for a control population of particles may thus allow for characterisation of the assayed population of particles accordingly, i.e. as being the same state as the control particles; or non-equivalent positions may characterise the assayed particles as not being ofthe same state as the control particles. The skilled person understands how to determine equivalence in this context, and appreciates that equivalent positions do not require complete overlap of contours, so long as they share sufficiently significant statistical nexus (e.g. the overlap can be at least 75%, at least 80%, at least 90% or at least 99%).

[0032] In a suitable embodiment, a method of the invention (for example a method of the first, second, third or fourth aspects of the invention) may further comprise determining the presence of a third, or subsequent marker, and characterising the state of the particle, or population of particles, on the basis of one or more comparisons between the determinations in respect of the first, second, third (and subsequent) markers. The third marker (and any subsequent marker or markers) may be a glycan marker, or a non-glycan marker, in keeping with the considerations set out below. A method of the invention may involve determining the presence of a single glycan marker and a single non-glycan marker, determining the presence of a plurality of glycan markers and a single non-glycan marker, determining the presence of a single glycan marker and a plurality of non-glycan markers, or determining the presence of a plurality of glycan markers and a plurality of non-glycan markers.

[0033] Similarly, a method of the invention (for example a method of the first, second, third or fourth aspects of the invention) may further comprise determining the presence of markers in respect of a third, or subsequent, particle or population of particles, and characterising the state of these particles, or population of particles, on the basis of one or more comparisons between the determinations in respect of the requisite markers.

[0034] In a suitable embodiment, a method of the invention may involve one or more determination undertaken in respect of a particle, and one or more determination undertaken in respect of a population of particles, and characterisation of the particle and / or population of particles on the basis of these determinations.

[0035] BRIEF DESCRIPTION OF THE FIGURES

[0036] Figure legends

[0037] For a better understanding of the invention, and to show how embodiments of the invention may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: -Figure 1 - Flow cytometry analysis of senescent markers on HUVEC cells after DOXO treatment.

[0038] HUVEC from three different donors, at passage 4 or 5, were pooled and subjected to DOXO treatment for senescence induction. Young and DOXO treated HUVEC were analyzed for beta-galactosidase activity with DDAO (A, B); p16 (C, D) and cell size with forward scatter (FSC) (E). Percentage positive DDAO or P16 cells are shown in histograms (A-D). Young (darker grey datapoints) and DOXO treated (lighter grey datapoints) HUVEC are shown in forward (FSC) vs side (SSC) scatter dot-plot (E). Geometric mean of forward scatter, FSC-A, is shown (E). An alternative view of (E) is shown in (F).

[0039] Figure 2 - Flow cytometry analysis of senescent markers on fibroblasts after DOXO treatment. Fibroblasts from three different donors, at passage 5, were pooled before DOXO induction of senescence. Young and DOXO treated fibroblasts were analyzed for betagalactosidase activity with DDAO (A, B) and cell size with FSC (C). Expression levels of DDAO in both young (A) and DOXO treated fibroblasts (B) are shown in histograms. Percentage positive DDAO cells are shown in histograms (A, B). Young (darker grey datapoints) and DOXO treated (lighter grey datapoints) fibroblasts are shown in forward (FSC) vs side (SSC) scatter dot-plot and their geometric mean of FSC-A, is shown (C). An alternative view of (C) is shown in (D).

[0040] Figure 3 - Flow cytometric analysis of senescence marker panel on MSC. Flow cytometry was used to confirm the establishment of senescence in MSC upon replicative- and induced-senescence. Levels of beta-galactosidase activity (A), p16 (B), yH2AX (C) and ki-67 (D) were analysed at early (P5), intermediate (‘Interim’ P11-13) and late (P16-25) passages, as well as after induction of senescence with DOXO. Values are expressed as mean ± SD of n=5 donors. One-Way Anova test was used for replicative senescence (early vs interim vs late passages) and unpaired, parametric student t-test for induced senescence (early vs DOXO-treated). Figure 4 - Luminex multiplex analysis of MSC SASP upon replicative- and induced-senescence. MSC secretion levels of IL-6 (A), IL-8 (B) and IGFBP-2 (C), all part of SASP, at early (P4-5), intermediate (‘Interim’ P11-13) and late (P16-25) passages, as well as after induction of senescence with DOXO. Values are expressed as mean ± SD of n=5 donors. One-Way Anova test was used for replicative senescence (early vs interim vs late passages) and unpaired, parametric student t-test for induced senescence (early vs DOXO-treated). Figure 5 - Percentage positive flow cytometry analysis of lectin binding to youngproliferating and DOXO treated primary cells. Flow cytometry analysis of lectin binding to three different cell types following DOXO treatment. (HUVEC: top, fibroblasts: middle, BMMSC: bottom), expressed as percentage positivity. Lectins used: MAL-II, SNA, NPL, GNA,AAL, UEA-I, STL, SJA and PNA. Three donors were pooled per cell type, Percentages as indicated.

[0041] Figure 6 - MFI flow cytometry analysis of lectin binding to young-proliferating and Doxo-treated primary cells. Flow cytometry analysis of lectin binding to three different cell types following DOXO treatment. (HUVEC: top, fibroblasts: middle, BMMSC: bottom), expressed as MFI. Three donors were pooled per cell type. MFI as indicated.

[0042] Figure 7 - Percentage positive flow cytometry analysis of lectin binding to DPMSC upon replicative senescence. Flow cytometry analysis of lectin binding to human DPMSC at early (P5) and late (P22-24) passage, expressed as percentage positive binding, n = 5 donors. Data expressed as means ± SD and unpaired, parametric student t-test was used.

[0043] Figure 8 - MFI flow cytometry analysis of lectin binding to DPMSC upon replicative senescence. Flow cytometry analysis of lectin binding to human DPMSC at early (P5) and late (P22-24) passage, expressed as MFI. n = 5 donors. Data expressed as means ± SD and unpaired, parametric student t-test was used.

[0044] Figure 9 - Percentage positive flow cytometry analysis of lectin binding to DOXO induced senescent DPMSC. Flow cytometry analysis of lectin binding to young-proliferating (P5) and DOXO-treated (P6) human DPMSC, presented as percentage positive. MAL-II, STL, SJA, UEA-I, AAL, GNA (n = 3 donors for each of the previous lectins); SNA (n = 6 donors); NPL (n = 1 donor). Data is expressed as means ± SD and unpaired, parametric student t-test was used.

[0045] Figure 10 - MFI of lectin panel flow analysis of DPMSC upon induction of senescence.

[0046] Lectin data of young-proliferating (P5) and Doxo-treated (P6) DPMSC is presented as MFI. MAL-II, STL, SJA, UEA-I, AAL, GNA(n = 3 donors for each of the previous lectins); SNA (n = 6 donors); NPL (n = 1 donor). Data is expressed as means ± SD and unpaired, parametric student t-test was used.

[0047] Figure 11 - Young and senescent cell purification by lectins. Young cells were stained with cell tracer violet and senescent cells (Doxo-treated) were stained with cell tracer red. These stained cells were mixed in 1:1 ratio. Cells were incubated with either individual lectins at different concentrations as indicated, or a mixture of lectins and subsequently bound to magnetic affinity beads prior to magnetic separation. Pre-separation cells (left-most bars) as well as flow-through cells (middle bars) and bound cells (right-most bars) were analyzed by flow cytometry. Only data from one round of purification was captured. Analysis was performed on HUVEC (A), fibroblasts (B) and BMMSC (C). The ratio of young to old cells in either the flow-through population or the bound population are expressed as a percentage relative tothat in the pre-sort population. Cells from three donors were pooled and used in a single experiment.

[0048] Figure 12 - Early fibroblast lectin changes after staurosporine treatment in Annexin V negative cells

[0049] 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 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 negative subpopulations are shown in (A and C).

[0050] Figure 13 - Early MSC lectin changes after staurosporine treatment in Annexin V negative cells

[0051] 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 timepoint is expressed as a percentage relative to that of the DMSO vehicle control. 100% dotted line indicate no change (A). Annexin 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). SJAand 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 percentage positive value is indicated for the whole population (F). Annexin negative subpopulations are shown in (A, C, D and E).

[0052] Figure 14- Early MSC lectin changes after treatment with anti-FAS antibody in Annexin V negative cells

[0053] 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, SNAand 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 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 negative subpopulations are shown in (A and C).Figure 15 - Early HUVEC (Human umbilical vein endothelial cells) lectin changes after staurosporine treatment in Annexin V negative cells

[0054] 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 (C). Annexin V negative subpopulations are shown in (A and C).

[0055] Figure 16 - Gating Strategy

[0056] 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).

[0057] All samples shown, unless otherwise specified, are from the Annexin negative gate.

[0058] Figure 17 - 2D Lectomics analysis reveal multiple stages of apoptosis

[0059] (A) Comparison of one-dimensional (PS only) and two-dimensional (Lectin + PS) flow analysis of apoptosis. Intrinsic apoptosis of MSC is induced by Staurosporine for 18h.

[0060] Above: analysis with Annexin-V histograms. Lighter grey data: DMSO, darker grey data: Staurosporine treatment for the same time periods. Below: GNA-PE and Annexin-V two-dimensional analysis of the same sample. Lowest contour(s): Secondary only control (Streptavidin-PE), Highest contour(s): GNA-PE. Overlaid contour plots applied. Apoptotic cell and body subpopulations are gated, based on 12h timepoint. M (mother cell population); M’ and M” (cells with increasing PS); Pop 4 (the first apoptotic body subpopulation), Pop 5 and Pop 6 (later emerging apoptotic body subpopulation with distinct lectin binding).

[0061] (B) 12thhour lectomics: all lectin binding displayed using Lectin + PS 2D flow analysis.

[0062] Lighter grey contours: secondary only control, darker grey contours: Lectin-PE (C) Lectin binding identifies the first emergence of apoptotic bodies. Left: Pie chart showing the emergence of Pop 4, the first apoptotic body subpopulation, in relation to the M, M’, M” subpopulations.

[0063] Figure 18 - graphical view of 10 stages of apoptosis. These technological advances allowed study of all ten stages of apoptosis simultaneously and permit quantitative comparisonof glycan expression across the apoptosis process. For each population, AAL = left most bar; GNA= middle bar; SNA= right most bar.

[0064] Figure 19 - Size profile of Lectomic Apoptotic stages

[0065] (A) Pie chart showing percentage of individual populations of cells and apoptotic bodies at the 12thhour after induction of intrinsic apoptosis by Staurosporine.

[0066] (B) Changes in the size of the M pop diameter as measured by FSC-H.

[0067] (C) Compiled population diameters as measured by FSC-H across all major populations from 1 h to 18h

[0068] (D) Pie charts showing percentage of each of six populations from 1h to 18h.

[0069] Figure 20 - An example of a sugar wheel legend. Each segment can vary in size (from the centre) to indicate glycan levels.

[0070] Figure 21 - Apoptotic to Necrotic transition.

[0071] Figure 22 - Apoptosis vesicle lectomics.

[0072] Figure 23 - comparing 1D and 2D analysis.

[0073] Figure 24 - comparing healthy / unhealthy cells.

[0074] Figure 25 - cell cycle monitoring. A - level changes in percentage; B - same data as the table in A, but translated into fold changes, expressed relative to CD8Dim cells in P0, G1 ; C -corresponding sugar wheels of data in A and B.

[0075] Figure 26 - Programme of sugar density fingerprint changes during T cell differentiation. PBMCs from healthy patient is subjected to Lectomic analysis. Monocytes are depleted leaving behind mostly B and T cells. CD4-CD8- negative is predominantly B cells. Sugar wheel presentation of lectin binding density relative to levels identified for B cells (CD4-CD8- in this case). Activation levels of CD8 and CD4 T cells are indicated by diminishing expression of CD8 and CD4 respectively. In the image, the quoted blue ring is the outer ring, and the quoted red ring is the inner ring.

[0076] Figure 27 -Traditional histograms of lectin binding across the Lectome panel.

[0077] Histograms indicating levels of lectin binding is displayed. For CD4 T cells, highest histogram: total CD4, second highest histogram: CD4 hi, second lowest histogram: CD4 dim, control is the lowest histogram. CD4-CD8-. For CD8 T cells, highest histogram: CD8 total, second highest histogram: CD8 hi, third highest histogram: CD8 dim, second lowest histogram: CD8 super dim, control is the lowest histogram. CD4-CD8-.Figure 28 - shows a schematic to illustrate the effect of moderation of flow cytometer data based on size.

[0078] Figure 29 - shows a box diagram of an example system of the invention.

[0079] Figure 30 - shows a flow diagram of an example method of the present invention.

[0080] Figure 31A - shows a plot of flow cytometry data without moderation by size for a specific sample. Fig. 31 B shows a plot of the same flow cytometry data of Fig. 31 A, wherein the data on the y-axis has been moderated according to the method of Fig. 30, based on the FSC signal. Fig. 31 C shows a plot of the same flow cytometry data of Fig. 31 A, wherein the data on the x-axis has been moderated according to the method of Fig. 30, based on the FSC signal. Fig. 31 D shows a plot of the same flow cytometry data of Fig. 31 A, wherein the data on the x-axis and the y-axis has been moderated according to the method of Fig. 30, based on the FSC signal.

[0081] Figure 32A - shows a plot of flow cytometry data without moderation by size for another specific sample; whilst Fig. 32B shows a plot of the same flow cytometry data of Fig. 32A, wherein the data on both axes has been moderated according to the method of Fig. 30. Figure 33 - shows two sets of comparative plots comparing flow cytometry data in both “raw”, unmoderated form, and moderated form according to the method of Fig. 30. Each set of comparative plots relates to a respective cell type, subjected to the same treatments at various time points.

[0082] Figure 34 - shows a subset of the data from Fig. 33 being subjected to confirmatory viability analysis. For example, with the bottom to graphs showing a working example of sDEN and comparison to vDEN viability dye.

[0083] Figure 35 - shows a flow diagram of another example method of the present invention. Figure 36 - shows a flow diagram of another example method of the present invention, for example wherein the method of Fig. 36 is an example of a method according to Fig. 35. Figure 37 - shows a contour plot with an overlaid grid matrix, having segments that each define a dimensional space. Central dimensional space is DS1 (arrow). The grid includes nine dimensional spaces, but can include more. Analysis based on eF780.DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention is based upon the inventors’ finding that glycan markers on the surface of cell membranes, whether used as part of a panel of glycan markers, or as part of a panel incorporating other marker types, provide previously unrecognised insights into the state of the particle (such as a cell or vesicle) to which the membrane belongs. This finding enables the use of the methods and kits set out in this disclosure, which may be used characterise such particles based on their biological state.

[0085] In particular, the inventors have found that changes in the glycan marker profile of cells may provide an earlier indication in changes of cell state than do previously recognised markers. Furthermore, the degree of sensitivity that can be achieved using the methods and kits of the invention can enable the identification of important sub-populations of cells or vesicles in populations that had previously been thought to be homogeneous.

[0086] As well as providing an insight into specific changes in biological states of interest, the methods and kits of the invention have very broad applicability, enabling identification of changes in state, and characterisation based on such changes, even when the stimuli or mechanisms underlying the causes of the changes are not known. As such, the methods and kits of the invention provide powerful new tools for the investigation of cell states in a very broad range of contexts, including, but not limited to diagnostic and prognostic applications, drug screening, and the ability to identify fundamental changes in cell behaviour (such as changes associated with programmed cell death or senescence).

[0087] The invention will now be further described with reference to various definitions which may be useful in practicing the invention, or in considering the scope of protection sought.

[0088] Except for where the context requires otherwise, definitions provided in respect of constituents (whether particles or reagents) to be used in the methods of the invention should also be taken as applicable to constituents of kits of the invention, and vice versa.

[0089] Where terms such as a terminal sialic residue “bindable by” MAL-II (e.g. the residue then being denoted to be a “MAL-II residue”), are used, this is intended to encompass methods that involve determining the terminal glycan (that can be bound by MAL-II) with alternative binding partners (additionally or alternatively to the actual lectin quoted e.g. MAL-II in the context of the example above).When referring to fold changes the term “about” is used e.g. “about 2.0 fold”. The term “about” preferably means that the fold change value can vary by + / - 10% (preferably by + / - 5%).

[0090] A particle comprising a cell membrane

[0091] For the purposes of the present disclosure, a reference to “a particle comprising a cell membrane” may be taken as referring to any particle made up entirely, or in part, of a biological cell membrane.

[0092] In a suitable embodiment, the particle is encompassed by the cell membrane, creating an exterior cell membrane surface of the particle, and an interior of the particle.

[0093] Suitable examples of such particles include cells, which may be from unicellular or multicellular sources. Live cells represent particularly suitable examples of particles that may be used. Other examples of such particles include fragments or components of such cells (including vesicles, organelles, and cell apparatus). Specific examples of particles comprising cell membranes that may advantageously be used in methods of the invention are considered elsewhere in the specification.

[0094] A population of particles comprising a cell membrane

[0095] In the context of the present invention, “a population” of particles comprising a cell membrane may be taken as being any plurality of particles meeting the requirements set out above.

[0096] A suitable population of particles may be a population of cells, or a population of vesicles. A suitable population of particles may be a mixed population of cells and vesicles.

[0097] A population may comprise a single form of particles. Such a population may be termed a homogeneous population. A suitable homogeneous population may be a homogeneous population of cells. Alternatively, a suitable homogeneous population may be a homogeneous population of vesicles.

[0098] A suitable population may comprise a plurality of different forms of particles. Such a population may be termed a heterogeneous population. A suitable heterogeneous population may be a heterogeneous population of cells. Alternatively, a suitable heterogeneous population may be a heterogeneous population of vesicles.Further details of embodiments of populations of particles comprising cell membranes that may usefully be employed in the methods of the invention are described further in connection with relevant embodiments of the invention below.

[0099] A marker

[0100] A marker, for the purposes of the present disclosure, may be taken as being any molecule or structure associated with a particle that can be detected, whether directly or indirectly, to provide information about the state of the particle (or about a population of such particles).

[0101] Suitable markers may be external markers (for example found associated with the external surface of a particle) or internal markers (for example found in the interior of a particle).

[0102] The methods of the invention make use of first and second markers, and may optionally further employ third or subsequent markers. Except for where context requires otherwise, disclosures in the present specification referring to first and second markers should also be considered to encompass methods or kits of the invention employing third markers and subsequent markers.

[0103] Methods of the invention may be directed to characterising the state of a particle within a particular biological pathway such as apoptosis (e.g. where the particles are putatively in given state of such biological pathway), and the second marker increases, decreases or does not change as a function of the progression state of said biological pathway. In other words, the second marker may demonstrate variability (or lack thereof) that is characteristic of the progression state of said biological pathway. An example is phosphatidyl serine (e.g. increasing as a function of the progression of the cell death pathway).

[0104] Determining the presence of a marker

[0105] Determination of the presence of a marker may be taken as encompassing any assessment that allows a conclusion to be drawn as to whether a marker is or is not present.

[0106] For the avoidance of doubt, a finding that a specified marker is not present will also be considered to meet the requirement for “determining the presence of a marker” (in that this finding constitutes a definitive negative determination of the presence of the marker in question).In a suitable embodiment, a determination as to the presence of a marker may be a binary determination (i.e. that the marker is or is not present). In another suitable embodiment, a determination as to the presence of a marker may be a quantitative determination (i.e. a determination that provides information as to the amount of the marker present).

[0107] Suitably, the presence of the first and second markers (and optionally the third or further marker where present) are detected simultaneously, and for an individual particle.

[0108] Thus, methods of the invention a preferably predicated on single-cell analysis, where the first and second markers (and optionally the third or further marker where present) each for an individual particle are used to characterise the state of said same individual particle. For methods that involve a population of particles, it is particularly preferred that presence of the first and second markers (and optionally the third or further marker where present) for each individual (assayed) particle is determined (e.g. simultaneously), wherein the first and second markers for each individual particle of the population is used to characterise the state of said same individual particle.

[0109] As will be discussed below, such single cell analysis can be achieved with flow cytometry.

[0110] Lectin binding partners

[0111] A lectin suitable for use as a binding partner for use in a method or kit of the invention may be selected from the group consisting of: aleuria aurantia lectin (AAL), maackia amurensis lectin II (MAL-II), sambucus nigra lectin (SNA), peanut agglutinin (PNA), styphnolobium japonicum (SJA), Galanthus nivalis lectin (GNA), narcissus pseudonarcissus lectin (NPL), solanum tuberosum lectin (STL), ulex Europaeus Agglutinin I (UEA-1), Lotus Tetragonolobus lectin (LTET), Ricinus communis agglutinin I (RCA), Anti-Tn Salvia Sclarea lectin (SSA), and Viscum album agglutinin I (VAA).

[0112] Characterising the state of a particle or population of particles

[0113] Characterisation of a state of a particle or population of particles may be taken as being any analysis providing information as to the state of the particle. For example, in the case of a particle that is a cell, characterisation may, without limitation, be used to provide information on the health state of the cell (for example whether the cell is uncompromised, apoptotic, senescent, or necrotic), a disease state of the cell, the cell cycle state (e.g. G phase state, S phase state etc) of the cell, the viability of the cell, or the developmental state of the cell.Suitably all of the particles within a given population may be characterised as having the same state. An example of such a situation may be a homogeneous population of cells.

[0114] Alternatively, particles within a given population may be characterised as having different states. Merely by way of example, such a situation may arise in the case of a heterogeneous population of cells. Such heterogeneous population of cells may all be of the same ‘type’ (e.g. all being mesenchymal stem cells, as an example), with the heterogeneity being provided by the existence of more than one cell state within the population.

[0115] In a suitable embodiment, a method in accordance with the first aspect of the invention may be used to characterise the state of a first and / or second particle, by comparing markers (at least first and second markers, but also optionally third or subsequent markers) associated with the first and second particles. In such an embodiment, the method may comprise: determining the presence of a glycan first marker on the external surface of a first particle; and determining the presence of a second marker associated with the first particle, as set out in the methods of the first aspect of the invention, and may comprise further steps of:

[0116] • determining the presence of the glycan first marker on the external surface of a second particle;

[0117] • determining the presence of the second marker associated with the second particle;

[0118] and

[0119] • comparing the results achieved in respect of the first and second markers in the first and second particles, and

[0120] characterising the state of the first and / or second particle on the basis of these determinations.

[0121] Indeed, so useful are methods in accordance with this embodiment, that they give rise to the methods of the third aspect of the invention (defined above, in the summary of the invention).

[0122] In a similar manner, a method in accordance with the second aspect of the invention may be used to characterise the state of a first and / or second population of particles, by comparing markers (at least first and second markers, but also optionally third or subsequent markers) associated with the first and second population of particles. In such an embodiment, the method may comprise: determining the presence of a glycan first marker on the external surface of particles within a first population; and determining the presence of a second marker associated with particles within a second population, as set out in the methods of the second aspect of the invention, and may comprise further steps of:• determining the presence of the glycan first marker on the external surface of particles associated with a second population;

[0123] • determining the presence of the second marker associated with particles of the second population; and

[0124] • comparing the results achieved in respect of the first and second markers in the first and second particles, and

[0125] characterising the state of the first and / or second population of particles on the basis of these determinations.

[0126] These advantages give rise to the methods of the fourth aspect of the invention (defined above, in the summary of the invention).

[0127] A method of the third or fourth aspect of the invention, or a corresponding embodiment of a method of the first or second aspects of the invention, may further comprise determining the presence of a third (or subsequent) marker in respect of the particles or populations of particles investigated. In connection with this, it is also worth noting that a method of the third or fourth aspect of the invention, or a corresponding embodiment of a method of the first or second aspects of the invention, may further comprise determining the presence of markers in third (or subsequent) particles, or third (or subsequent) populations of particles.

[0128] In a method of the third or fourth aspect of the invention, or corresponding embodiments of the methods of the first or second aspects of the invention, the first and second particles, or populations of particles, may be cells, or vesicles, or populations of cells or vesicles. Suitable cells or vesicles, or populations thereof, may be selected in order to investigate differences between the cells or vesicles (or populations of cells and / or vesicles) of interest.

[0129] Except for where context requires otherwise, one of the first or second particles, or populations of particles, may be selected to be a suitable control or comparator to be used with particles, or populations of particles, of interest. Alternatively, both first and second (and optionally third and subsequent) particles, or populations of particles, may be selected on the basis that they are of interest, and therefore suitable for investigation.

[0130] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from healthy and diseased sources.In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources at different subdivisions ora biological pathway. In a suitable example of such an embodiment, the sources may be at different stages of apoptosis.

[0131] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources at different subdivisions in the progression of a disease. In a suitable example of such an embodiment, the sources may be at different clinical stages of a disease, such as cancer.

[0132] Methods of the invention in accordance with embodiments of the sort set out in the preceding paragraphs may have applications in diagnostic contexts.

[0133] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from diseased sources when exposed to known or putative treatments for the disease. Such methods may be used for drug screening. Suitably such methods may be adapted for high throughput screening applications.

[0134] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources at different stages in a disease’s response to treatment. In an example of this sort, the cells, or populations of cells, may be selected to provide a time course of the response (or lack of response) exhibited by the disease.

[0135] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from different sources having the same disease. For example, the cells, or populations of cells, may be derived from different subjects having the same disease. In such an embodiment, the methods may be used to characterise the cells from the different subjects, thus allowing comparison of the way in which different subjects respond to a disease.

[0136] It will be appreciated that methods of the invention in accordance with embodiments of the sort set out in the preceding paragraphs may have applications in screening and development of therapeutic agents, or the development of treatment regimens.

[0137] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources at different stages in the progression of a disease. For example, the cells, or populations of cells, may be selected to provide a timecourse of the progression of the disease. Suitably, the cells, or population of cells, may be selected to provide a time course of the progression of the disease in the absence of treatment. Information gained regarding the state of such untreated cells, or populations of cells, may be compared with information regarding the state of comparable cells undergoing treatment. For example, the comparable cells undergoing treatment may be selected on the basis of their positive response to treatment. Comparison in such an embodiment may be used to identify how treatment intervenes in the progression of disease to bring about effective therapy. Alternatively, the comparable cells undergoing treatment may be selected on the basis of their poor response to treatment. Comparison in such an embodiment may be used to identify how potential treatments fail to provide effective therapy to certain patients.

[0138] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources identified as having genetic alterations associated with a disease. For example, such embodiments may be used to characterise the states of cells from sources having mutations known to be associated with a disease. Suitably, such mutation may be associated with the development of a disease, or with the response of a disease to treatment.

[0139] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells from sources identified as having metabolic changes associated with a disease, or associated with an environmental condition (e.g. a subject’s diet). For example, such embodiments may be used to characterise the states of cells from sources metabolic changes associated with the development of a disease, or with the response of a disease to treatment.

[0140] It will be appreciated that methods of the invention in accordance with embodiments of the sort set out in the preceding paragraphs may have applications in prognostic methods. For example, such methods may be useful in indicating the likely outcome of a subject’s disease once a diagnosis has been made.

[0141] The methods of the invention are also highly applicable to use in investigating the states of cells used in in vitro or in vivo models.

[0142] In a suitable embodiment, different first and second cells, or populations of cells, may be used to characterise the states of cells that have been subject to modification or gene editing. For example, suitable cells, or populations of cells, may have been subject to gene knockout, or to gene knock in. Suitable cells may have been subject to gene editing using a techniquesuch as CRISPR. The methods may be practiced using cells, or populations of cells, subject to natural or induced epigenetic changes. The methods may be practiced using cells, or populations of cells, comprising CRISPR arrays.

[0143] In a suitable embodiment, a method of the invention may involve characterising the state of a particle, or of a population of particles, in a number of instances. The inventors have found that methods in accordance with this embodiment enable particularly useful analysis of changes in the particles or populations in novel and advantageous ways.

[0144] Suitably, such a method involves determining the presence of first and second markers on first and second instances, as set out below. Such a method may also involve determining the presence of third (or subsequent) markers, and additionally, or alternatively, may involve determining the presence of a required number of markers on third (or subsequent) instances.

[0145] The determinations made in respect of the required markers on the first, second, and optionally third (and subsequent), instances allow a time series of results to be established. Analysis of any changes in determinations of marker presence (e.g. which may refer to its level as discussed elsewhere herein) with time can provide further useful information in the characterisation of particles, or populations of particles.

[0146] In a suitable embodiment, a method according to the first aspect of the invention may comprise characterising the state of a particle, in a method further comprising:

[0147] • determining the presence of a glycan first marker on the surface of the particle in a first instance and in a second instance;

[0148] • determining the presence of a second marker associated with the particle in a first instance and in a second instance;

[0149] • comparing the results achieved in respect of the first and second markers in the first and second instances.

[0150] In a suitable embodiment, a method according to the second aspect of the invention may comprise characterising the state of a population of particles, in a method further comprising:

[0151] • determining the presence of a glycan first marker on the surface of the particles within a population in a first instance and in a second instance;

[0152] • determining the presence of a second marker associated with particles within a population in a first instance and in a second instance;comparing the results achieved in respect of the first and second markers in the first and second instances.

[0153] In a suitable embodiment, a method according to the third aspect of the invention may comprise characterising the state of a first and / or second particle, in a method further comprising:

[0154] • determining the presence of the glycan first marker on the surface of the first particle in a first instance and in a second instance;

[0155] • determining the presence of the second marker associated with the first particle in a first instance and a second instance;

[0156] • determining the presence of the glycan first marker on the surface of the second particle in a first instance and in a second instance;

[0157] • determining the presence of the second marker associated with the second particle in a first instance and a second instance;

[0158] • comparing the results achieved in respect of the first and second markers, for the first and second particles, in the first and second instances.

[0159] In a suitable embodiment, a method according to the fourth aspect of the invention may may comprise characterising the state of a first and / or second population of particles, in a method further comprising:

[0160] • determining the presence of the glycan first marker on the surface of the particles within the first population in a first instance and in a second instance;

[0161] • determining the presence of the second marker associated with particles within the first population in a first instance and a second instance;

[0162] • determining the presence of the glycan first marker on the surface of the particles within the second population in a first instance and in a second instance;

[0163] • determining the presence of the second marker associated with the particles within the second population in a first instance and a second instance;

[0164] • comparing the results achieved in respect of the first and second markers, for the first and second populations, in the first and second instances.

[0165] As previously mentioned, the glycan markers may be determined on the external surface of a particle, or of particles in a population.

[0166] In a suitable embodiment, the determination made in the first instance may be made in respect of a first particle, and the determination made in the second instance is made in respect of asecond particle. In a suitable embodiment, the determination made in the first instance may be made in respect of a first population of particles, and the determination made in the second instance may be made in respect of a second population of particles.

[0167] In a suitable example of an embodiment in which the determination in respect of the first and second markers is made on different particles, the particles are cells. In a suitable embodiment the cells are cells of the same population.

[0168] In a suitable embodiment, the determination in respect of the first and second markers is made in respect of a population of cells that are synchronised with respect to their cell cycles. Suitably, the determination in respect of the first and second markers is made in respect of a population of cells that are not synchronised with respect to their cell cycles.

[0169] Suitably, the first and second instances may be separated by a selected period of time. As discussed further elsewhere, suitably a stimulus is applied to the particle, or population of particles, during the time between the first and second instances. Alternatively, or additionally, a stimulus may be applied to the particle, or population of particles, prior to the first instance.

[0170] In a suitable embodiment, the comparison is performed in respect of results achieved at a single time-point.

[0171] In a suitable embodiment, the comparison is performed in respect of results achieved at a plurality of time-points.

[0172] In a suitable embodiment, the comparison is performed in respect of compressed data from the plurality of time-points.

[0173] In a suitable embodiment, the comparison comprises analysing a 2D plot of the results. For example, suitably the comparison comprises analysing a gradient in a 2D plot of the results.

[0174] Methods of each of the broadest aspects of the invention, as described in the summary of the invention above, may involve analysing a 2D plot generated based on the determination of the presence of the first and second markers, and characterising the state of the particle on the basis of this analysis.A 2D plot may plot a level of the first glycan marker for a particle, on a first axis (e.g. Y-axis), as a function of a level of the second marker for said same particle (e.g. the latter being plotted on a second axis, e.g. X-axis).

[0175] Said level (for a marker described herein) may be represented by an intensity determined for a detection moiety associated with a binding partner for a marker described herein. For example, said level of intensity may be a fluorescence intensity such as a mean fluorescence intensity, more particularly for embodiments described elsewhere herein that involve a binding partner, for the first and second markers (and optionally subsequent marker), the binding partner being labelled with a detection moiety that comprises a fluorophore.

[0176] A 2D plot may be generated as a step of a method of the invention e.g. prior to a step of charactering the state of a particle. Thus, a 2D data-point for an individual particle, or for a subset of particles or for each individual particle of a population of particles, may be generated; and methods of the invention may involve characterising the state of the particle (or each particle) on the basis of this 2D data-point (e.g. on the basis of its position in 2D space).

[0177] The advantages of such 2D plot are numerous, as will be outlined below.

[0178] As mentioned above, a 2D data-point for an individual particle may be compared with a corresponding 2D data-point for a particle of known state, and the state of the particle may be determined on basis of this comparison.

[0179] For methods involving a population of particles, a 2D data-point for each assayed particle may be plotted on a single 2D plot, which may therefore demonstrate the presence of a homogenous population of particles having the state, or the presence of a heterogenous population of particles having different states.

[0180] Suitably, for methods involving a population of particles, a method may comprise generating a contour plot based on the distribution of data-points on the 2D plot. The skilled person understands how to generate a contour plot (e.g. in flow cytometry), which visualizes cell populations as density maps using contour lines, similar to a topographical map, where closer, darker lines indicate higher particle (as an event) concentration, making it easier to detect distinct groups than with basic dot plots, especially for complex data or identifying boundaries for gating.More particularly, the generation of such contour may involve establishing a contour on the 2D plot that indicates a boundary of the distribution of a population of similar particles [of the same state], wherein the boundary encloses a central proportion of the data points (e.g. around the peak of the contour at the centre), wherein the proportion is 99%, 95%, 90%, 75%, or 50% such that outlying measurements are excluded.

[0181] Thus, a given “contour” may enclose each data-point for a particle of a given states, aiding the identification of a population of particles having said state (and the identification of the homogeneity or heterogeneity of states of the assayed particles).

[0182] Such contour plots may be particular advantageous as part of a cell sorting step (the latter being described elsewhere herein). For example, particles whose 2D data-points are encompassed by a defined contour (e.g. as a ‘gate’) may be selectively sorted / collected, optionally for further / downstream analysis as described elsewhere herein.

[0183] The presence of a single (defined) contour on a contour plot may be indicative of the presence of a homogenous population of particles (i.e. of the same state) in an assayed sample. Alternatively, the presence two or more contours may indicate the presence of a heterologous population of states (i.e. of different states). For example, analysis of a contour plot may allow for the identification of a pattern of contours associated with a particular biological pathway, and the state of any given particle may be determined based on which contour said particle (more particularly its 2D data point) falls under.

[0184] A 2D plot or a contour plot may be overlaid with grid e.g. where each segment of the grid defines a distinct 2D dimensional space. The state of a particle may be characterised based on which segment of the grid said particle (more particularly its 2D data point or associated contour) falls within.

[0185] In embodiments that involve determining the presence of markers of the invention at a first instance and at a second instance, a 2D plot or a contour plot may be provided for each of the first and second (and optionally any subsequent) instances. The 2D plot or contour plot for each instance may be compared. For example, the position of a particle (more particularly its 2D data point or associated contour) on a graph for a second instance may be compared with the position of said same particle (more particularly its 2D data point or associated contour) on a graph for the first instance. A difference in the position (e.g. dimensional space change) of the particle on the plot for the second instance, relative to its position on the plot for the first instance, may indicate a change in particle state between the first and second instances.The above-mentioned ‘grid’ may be particularly advantageous for such embodiments, as movement of a particle (more particularly its 2D data point or associated contour) to a different segment of the plot for the second instance, relative to its segment position on the plot for the first instance, may indicated a change in particle state between the first and second instances.

[0186] Marker correlations

[0187] A suitable embodiment of the methods described above may further comprise characterising the particle or population of particles as:

[0188] • a particle, or population of particles, in which presence of the first marker and presence of the second marker are correlated with one another; or

[0189] • a particle, or population of particles, in which presence of the first marker and presence of the second marker are not correlated with one another.

[0190] In a suitable embodiment of such a method, the particle or population is characterised as a particle or population in which presence of the first marker and presence of the second marker are correlated with one another, and in which the presence of the first marker and presence of the second marker are positively correlated with one another.

[0191] Alternatively in such a method, the particle or population may be characterised as a particle or population in which presence of the first marker and presence of the second marker are correlated with one another, and in which the presence of the first marker and presence of the second marker are negatively correlated with one another.

[0192] In a suitable embodiment, the first and second marker are correlated with each other in a linear manner. In an alternative embodiment, the first and second marker are correlated with each other in a non-linear manner.

[0193] In another suitable embodiment of a method as described above, the particle or population is characterised as a particle or population in which presence of the first marker and presence of the second marker are not correlated with one another, wherein the presence of the first marker is variable, and the presence of the second marker does not vary.

[0194] The inventors have found that embodiments of this sort may offer particular advantages in practice. In particular, and as demonstrated in the Examples below, the inventors have found that in embodiments of this sort, the methods of the invention may be able to confer a degreeof sensitivity that may not be possible using methods that characterise the state of a particle with reference to one or more prior art markers. When the presence of the glycan marker varies, but the presence of the second marker (which may be a prior art marker associated with a known cell state) does not vary, assessing cell state on the basis of the glycan marker (or markers) provide a method with improved sensitivity. This has been demonstrated in the Examples, in which phosphatidylserine (a marker the display of which is known to be associated with apoptosis among cells or vesicles) does not vary, while glycan markers do. In these embodiments, focusing cell state analysis on the glycan marker can provide an earlier indication that a cell or vesicle is apoptotic (particularly that the cell or vesicle is at a very early stage of apoptosis) than can be achieved focusing on phosphatidylserine instead.

[0195] In a suitable embodiment of such a method, the particle or population is characterised as a particle or population in which presence of the first marker and presence of the second marker are not correlated with one another, wherein the presence of the first marker does not vary, and the presence of the second marker is variable.

[0196] In a suitable embodiment of a method of the invention, the first and second instances are separated by a period of time.

[0197] Suitably, an investigative agent of interest is provided to the particles, or populations of particles, prior to the first instance. Alternatively, or additionally, an investigative agent of interest may be provided to the particles, or populations of particles, between the first instance and the second instance.

[0198] In a suitable embodiment, the investigative agent of interest is an inducer of a biological pathway. For example, the investigative agent of interest may be an inducer of apoptosis. In a suitable example, such an inducer of apoptosis is staurosporine.

[0199] In a suitable embodiment, the investigative agent of interest is a therapeutic agent, or putative therapeutic agent.

[0200] Glycan first markers

[0201] In a suitable embodiment of a method of the invention, the first marker is a glycan marker selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; an N-acetylgalactosamine (GalNAc) residue; a fucose residue; an N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.In a suitable embodiment of a method of the invention, the glycan first marker is a terminal glycan residue selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; and a terminal N-acetylglucosamine (GIcNAc) residue.

[0202] In a suitable embodiment the glycan first marker is terminal sialic acid residue, or a sialic acid residue at the -1 position immediately preceding the terminal glycan residue.

[0203] In a suitable embodiment the sialic acid residue is selected from the group consisting of: an alpha-2, 3-linked sialic acid residue (such as terminal NeuAc a2-3 Gal b1-3 GalNAc, which may be referred to as a residue bindable by MAL-II); and an alpha-2, 6-linked sialic acid residue (such as terminal NeuAc a2-6 Gal b1-4 GIcNAc residue, which may be referred to as a residue bindable by SNA).

[0204] In a suitable embodiment the glycan first marker is a terminal galactose residue, or a galactose residue at the -1 position immediately preceding the terminal glycan residue.

[0205] In a suitable embodiment, a terminal galactose is a galactosyl ((3-1 ,3) N-acetylgalactosamine (such as Fuc a1-2( Gal b1 -3 GalNAc a1-3) Gal b1-4 GIcNAc, which may be referred to as a residue bindable by PNA).

[0206] In a suitable embodiment, a terminal galactose is Fuc a1-3( Fuc a1-2( Gal a1-3) Gal b1-4) GIcNAc, which may be referred to as a residue bindable by SJA (preferably the B-SJA-I subunit).

[0207] In a suitable embodiment the glycan first marker is a terminal mannose residue, or a mannose residue at the -1 position immediately preceding the terminal glycan.

[0208] In a suitable embodiment, a terminal mannose is an alpha-1 , 3-linked mannose (such as alpha-1, 3-linked Man3GlcNAc2, which may be referred to as a residue bindable by GNA).

[0209] In a suitable embodiment, a terminal mannose is an alpha-1 , 6-linked mannose (such as alpha-1, 6-linked Man3GlcNAc2 or alpha-1 , 6-linked Man5GlcNAc2, which may be referred to as residues bindable by NPL).In a suitable embodiment the glycan first marker is a terminal N-acetylgalactosamine (GalNAc) residue, or a GalNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0210] In a suitable embodiment the glycan first marker is a terminal fucose residue, or a fucose residue at the -1 position immediately preceding the terminal glycan residue.

[0211] In a suitable embodiment, a terminal fucose is an a(1— >2) linked fucose, an a(1— >3), an a(1— >4) fucose linked fucose, more preferably an a(1— >6) linked fucose. An a(1— >6) linked fucose may be Fuc (a1,6) GIcNAc, which may be referred to as a residue bindable byAAL. An a(1— >2) linked fucose may be Fuc (a1,2) Gal (pi ,3) GIcNAc or Fuc (a1,2) Gal (pi ,4), which may be referred to as residues bindable by UEA-1.

[0212] In a suitable embodiment the glycan first marker is a terminal N-acetylglucosamine (GIcNAc) residue, or a GIcNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0213] In a suitable embodiment the glycan first marker is an internal N-acetyllactosamine residue.

[0214] In a suitable embodiment, an internal N-acetyllactosamine residue may be a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 bonds, which may be referred to as a residue bindable by STL.

[0215] Suitably, presence of the glycan first marker is determined by assessing binding of a binding partner for the first glycan marker.

[0216] Binding partners for glycan markers

[0217] In a suitable embodiment, a method of the invention comprises incubating the particle, or population of particles, with a binding partner for the first glycan marker. Suitably, the binding partner is selected from the group consisting of: a lectin; an antibody, or an antigen-binding fragment thereof; an aptamer; and a reporter cell. For example, the binding partner may be a lectin that binds to the glycan first marker.

[0218] In a suitable embodiment the glycan first marker is a sialic acid residue, and the binding lectin partner is selected from the group consisting of: MAL-II, and SNA.In a suitable embodiment the glycan first marker is a galactose residue, and the binding lectin partner is selected from the group consisting of: PNA, and SJA.

[0219] In a suitable embodiment the glycan first marker is a mannose residue, and the binding lectin partner is selected from the group consisting of: GNA, and NPL.

[0220] In a suitable embodiment the glycan first marker is a GalNAc residue, and the binding lectin partner binds to GalNAc.

[0221] In a suitable embodiment the glycan first marker is a fucose residue, and the binding lectin partner is selected from the group consisting of: AAL, UEA-1 , and LTET.

[0222] In a suitable embodiment the glycan first marker is a GIcNAc residue, and the binding lectin partner is G.Simp.

[0223] In a suitable embodiment the glycan first marker is an N-acetyllactosamine residue, and the binding lectin partner is STL.

[0224] Suitably the lectin is a recombinant lectin.

[0225] Suitably the lectin is an animal lectin. For example, the lectin may be a human lectin.

[0226] The lectin may be a plant lectin.

[0227] In a suitable embodiment, the particle or population of particles is animal derived (such as human derived) and the binding partner is derived from a non-animal source (for example a plant lectin). In a suitable embodiment, the particle or population of particles is human derived and the binding partner is derived from a non-human source (for example a non-human lectin, such as a mouse lectin, or a plant lectin). In a suitable embodiment, the particle or population of particles is plant derived and the binding partner is derived from a non-plant source (for example an animal lectin, such as a human or murine lectin).

[0228] In a suitable embodiment the binding partner is an antibody, or an antigen-binding fragment thereof, that binds to the glycan first marker. For example, the antibody, or antigen-binding fragment thereof, may be selected from the group consisting of: a monoclonal antibody; an ScFv antibody fragment; a recombinant Fc fusion; and a camelid antibody. In a suitable embodiment the antibody, or antigen-binding fragment thereof, binds to mannose.In a suitable embodiment the binding partner is a reporter cell. Suitably, a reporter cell expresses an agent that binds to a marker, such as a glycan first marker. It will be appreciated that a suitable agent to be expressed by a reporter cell may be selected with reference to the nature of the marker to be bound. For example, in the case of a reporter cell to bind to a glycan marker, the cell may express a glycan-binding lectin, or a glycan-binding antibody, or a glycan binding fragment thereof.

[0229] Methods using multiple glycan markers

[0230] Second glycan marker

[0231] In a suitable embodiment of any aspect of the invention, the second marker may be a glycan marker.

[0232] For the avoidance of doubt, it is preferred that the second marker is different to the first marker. Thus, while the second marker may be a second glycan marker (e.g. as an external marker), such embodiments therefore involve the determination of at least two different (i.e. first and second) glycan markers. In embodiments that involve determination of third or subsequent markers, it is likewise preferred that each marker is different, which may thus provide for three different glycan markers (where the second and third markers are also glycans).

[0233] Suitably, the second marker is a glycan marker selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; an N-acetylgalactosamine (GalNAc) residue; a fucose residue; an N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.

[0234] In a suitable embodiment the second marker is a terminal glycan residue selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; and a terminal N-acetylglucosamine (GIcNAc) residue.

[0235] In a suitable embodiment the second marker is terminal sialic acid residue, or a sialic acid residue at the -1 position immediately preceding the terminal glycan residue.

[0236] In a suitable embodiment the sialic acid residue (second marker) is selected from the group consisting of: an alpha-2, 3-linked sialic acid residue (such as terminal NeuAc a2-3 Gal b1 -3GalNAc, which may be referred to as a residue bindable by MAL-II); and an alpha-2, 6-linked sialic acid residue (such as terminal NeuAc a2-6 Gal b1-4 GIcNAc residue, which may be referred to as a residue bindable by SNA).

[0237] In a suitable embodiment the second marker is a terminal galactose residue, or a galactose residue at the -1 position immediately preceding the terminal glycan residue.

[0238] In a suitable embodiment, a terminal galactose (second marker) is a galactosyl ((3-1 ,3) N-acetylgalactosamine (such as Fuc a1-2( Gal b1 -3 GalNAc a1-3) Gal b1-4 GIcNAc, which may be referred to as a residue bindable by PNA).

[0239] In a suitable embodiment, a terminal galactose (second marker) is Fuc a1-3( Fuc a1-2( Gal a1-3) Gal b1-4) GIcNAc, which may be referred to as a residue bindable by SJA (preferably the B-SJA-I subunit).

[0240] In a suitable embodiment the second marker is a terminal mannose residue, or a mannose residue at the -1 position immediately preceding the terminal glycan.

[0241] In a suitable embodiment, a terminal mannose (second marker) is an alpha-1, 3-linked mannose (such as alpha-1, 3-linked Man3GlcNAc2, which may be referred to as a residue bindable by GNA).

[0242] In a suitable embodiment, a terminal mannose (second marker) is an alpha-1, 6-linked mannose (such as alpha-1, 6-linked Man3GlcNAc2 or alpha-1, 6-linked Man5GlcNAc2, which may be referred to as residues bindable by NPL).

[0243] In a suitable embodiment the second marker is a terminal N-acetylgalactosamine (GalNAc) residue, or a GalNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0244] In a suitable embodiment the second marker is a terminal fucose residue, or a fucose residue at the -1 position immediately preceding the terminal glycan residue.

[0245] In a suitable embodiment, a terminal fucose (second marker) is an a(1— >2) linked fucose, an a(1— >3), an a(1— >4) fucose linked fucose, more preferably an a(1— >6) linked fucose. An a(1— >6) linked fucose may be Fuc (a1,6) GIcNAc, which may be referred to as a residuebindable byAAL. An a(1— >2) linked fucose may be Fuc (a1,2) Gal (pi ,3) GIcNAc or Fuc (a1,2) Gal (pi ,4), which may be referred to as residues bindable by UEA-1.

[0246] In a suitable embodiment the second marker is a terminal N-acetylglucosamine (GIcNAc) residue, or a GIcNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0247] In a suitable embodiment the second marker is an internal N-acetyllactosamine residue.

[0248] In a suitable embodiment, an internal N-acetyllactosamine residue (second marker) may be a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 bonds, which may be referred to as a residue bindable by STL.

[0249] Suitably, presence of the second marker (e.g. as a glycan second marker) is determined by assessing binding of a binding partner for the second (e.g. glycan) marker.

[0250] Third or subsequent glycan marker

[0251] In a suitable embodiment of any aspect of the invention, the third or subsequent marker may be a glycan marker.

[0252] Suitably, the third or subsequent marker is a glycan marker selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; an N-acetylgalactosamine (GalNAc) residue; a fucose residue; an N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.

[0253] In a suitable embodiment the third or subsequent marker is a terminal glycan residue selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; and a terminal N-acetylglucosamine (GIcNAc) residue.

[0254] In a suitable embodiment the second marker is terminal sialic acid residue, or a sialic acid residue at the -1 position immediately preceding the terminal glycan residue.

[0255] In a suitable embodiment the sialic acid residue (third or subsequent marker) is selected from the group consisting of: an alpha-2, 3-linked sialic acid residue (such as terminal NeuAc a2-3 Gal b1-3 GalNAc, which may be referred to as a residue bindable by MAL-II); and an alpha-2, 6-linked sialic acid residue (such as terminal NeuAc a2-6 Gal b1-4 GIcNAc residue, which may be referred to as a residue bindable by SNA).

[0256] In a suitable embodiment the third or subsequent marker is a terminal galactose residue, or a galactose residue at the -1 position immediately preceding the terminal glycan residue.

[0257] In a suitable embodiment, a terminal galactose (third or subsequent marker) is a galactosyl (P-1,3) N-acetylgalactosamine (such as Fuc a1-2( Gal b1-3 GalNAc a1-3) Gal b1-4 GIcNAc, which may be referred to as a residue bindable by PNA).

[0258] In a suitable embodiment, a terminal galactose (third or subsequent marker) is Fuc a1-3( Fuc a1-2( Gal a1-3) Gal b1-4) GIcNAc, which may be referred to as a residue bindable by SJA (preferably the B-SJA-I subunit).

[0259] In a suitable embodiment the third or subsequent marker is a terminal mannose residue, or a mannose residue at the -1 position immediately preceding the terminal glycan.

[0260] In a suitable embodiment, a terminal mannose (third or subsequent marker) is an alpha-1, 3-linked mannose (such as alpha-1, 3-linked Man3GlcNAc2, which may be referred to as a residue bindable by GNA).

[0261] In a suitable embodiment, a terminal mannose (third or subsequent marker) is an alpha-1, 6-linked mannose (such as alpha-1, 6-linked Man3GlcNAc2 or alpha-1, 6-linked Man5GlcNAc2, which may be referred to as residues bindable by NPL).

[0262] In a suitable embodiment the third or subsequent marker is a terminal N-acetylgalactosamine (GalNAc) residue, or a GalNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0263] In a suitable embodiment the third or subsequent marker is a terminal fucose residue, or a fucose residue at the -1 position immediately preceding the terminal glycan residue.

[0264] In a suitable embodiment, a terminal fucose (second marker) is an a(1— >2) linked fucose, an a(1— >3), an a(1— >4) fucose linked fucose, more preferably an a(1— >6) linked fucose. An a(1— >6) linked fucose may be Fuc (a1,6) GIcNAc, which may be referred to as a residue bindable byAAL. An a(1— >2) linked fucose may be Fuc (a1,2) Gal (pi ,3) GIcNAc or Fuc (a1,2) Gal (pi ,4), which may be referred to as residues bindable by UEA-1.In a suitable embodiment the third or subsequent marker is a terminal N-acetylglucosamine (GIcNAc) residue, or a GIcNAc residue at the -1 position immediately preceding the terminal glycan residue.

[0265] In a suitable embodiment the third or subsequent marker is an internal N-acetyllactosamine residue.

[0266] In a suitable embodiment, an internal N-acetyllactosamine residue (third or subsequent marker) may be a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by p-1 ,4 bonds, which may be referred to as a residue bindable by STL.

[0267] Suitably, presence of the third or subsequent marker (e.g. as a glycan second marker) is determined by assessing binding of a binding partner for the third or subsequent (e.g. glycan) marker.

[0268] Generic description of glycans

[0269] Aterminal fucose (whether providing a first glycan marker, second marker or third / subsequent marker) 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 (a1 ,2) Gal (pi ,3) GIcNAc and / or Fuc (a1 ,2) Gal (pi ,4), with Fuc (a1 ,2) Gal (pi ,3) GIcNAc 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 fora terminal fucose, such as an a(1— >2) linked fucose linked (e.g. directly linked) to galactose (e.g. Fuc (a1,2) Gal (pi ,3) GIcNAc and / or Fuc (a1,2) 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 (a1,2) Gal (pi ,3) GIcNAc may also be presented as Fuc a1-2 Gal b1 -3 GIcNAc; the term Fuc (a1 ,2) Gal (pi ,4) may also be presented as Fuca1-2 Gal b1-4 GIcNAc; the term a(1— >2) linked fucose may also be presented as Fuc a1-2.

[0270] 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 a1-2” can be used to “a(1— >2) linked fucose” for convenience, and so on. Such alternative presentations may be used herein.Aterminal fucose (whether providing a first glycan marker, second marker or third / subsequent marker) 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 (GIcNAc), e.g. a GIcNActhat is more internal relative to said terminal fucose). More preferably, a terminal fucose may be an a(1— >6) fucose, linked to N-acetylglucosamine (GIcNAc)). Some examples include Fuc (a1 ,6) GIcNAc and Fuc a1-2 Gal [6S] b1-4 Glc; with Fuc (a1 ,6) GIcNAc 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 (GIcNAc), e.g. a GIcNAc 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 GIcNAc 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 a1-6, Fuc a1-2 and Fuc a1-3 (respectively); the term Fuc (a1 ,6) GIcNAc may also be presented as Fuc a 1-6 GIcNAc.

[0271] A terminal sialic acid (whether providing a first glycan marker, second marker or third / subsequent marker) may be an alpha-2, 3-linked sialic acid, preferably a terminal NeuAc a2-3 Gal b1 -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 b1 -3 GalNAc residue) may be MAL-II.

[0272] A terminal sialic acid (whether providing a first glycan marker, second marker or third / subsequent marker) may be an alpha-2, 6-linked sialic acid, preferably a terminal NeuAc a2-6 Gal b1-4 GIcNAc 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 b1-4 GIcNAc residue) may be SNA.

[0273] Aterminal N-acetylglucosamine (e.g. P(1 ,4)-linked GIcNAc) (whether providing a first glycan marker, second marker or third / subsequent marker) may be a monomer of N-acetylglucosamine and / or an oligomer of N-acetylglucosamine, such as Chitin and / or (GlcNAc)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(1 ,4)-linked GIcNAc) may be STL.An internal N-acetyllactosamine (e.g. LacNAc) (whether providing a first glycan marker, second marker or third / subsequent marker) described herein may be referred to as a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 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.

[0274] A terminal mannose (whether providing a first glycan marker, second marker or third / subsequent marker) may be a terminal alpha-1, 6-linked mannose, preferably alpha-1, 6-linked Man3GlcNAc2and / 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 Man3GlcNAc2and / or alpha-1, 6-linked Man5GlcNAc2) may be NPL.

[0275] A terminal mannose (whether providing a first glycan marker, second marker or third / subsequent marker) 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.

[0276] Throughout this specification, it is preferred that a “terminal mannose” is Man3GlcNAc2 and / or Man5GlcNAc2 (e.g. detectable via NPL / GNA lectins).

[0277] A terminal galactose (whether providing a first glycan marker, second marker or third / subsequent marker) may be a galactosyl ((3-1,3) N-acetylgalactosamine, for example, Fuc a1-2( Gal b1 -3 GalNAc a1-3) Gal b1-4 GIcNAc. 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 ((3-1,3) N-acetylgalactosamine, for example, Fuc a1-2( Gal b1 -3 GalNAc a1-3) Gal b1-4 GIcNAc) may be PNA.A terminal galactose (whether providing a first glycan marker, second marker or third / subsequent marker) may be Fuc a1-3( Fuc a1-2( Gal a1-3) Gal b1-4) GIcNAc, 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 a1-3 (Fuc a1-2 (Gal a1-3) Gal b1-4) GIcNAc) may be SJA.

[0278] A terminal N-acetylgalactosamine (whether providing a first glycan marker, second marker or third / subsequent marker) may be GalNAc b1-4 GIcNAc b1-3 GalNAc b1-4 GIcNAc. 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 b1-4 GIcNAc b1 -3 GalNAc b1-4 GIcNAc) may be SJA.

[0279] 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 ofSJA(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.

[0280] 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:

[0281] - GalNAc b1-4 GIcNAc b1-3 GalNAc b1-4 GIcNAc; this is referred to herein as a “terminal GIcNac”, e.g. with a GIcNac 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;

[0282] Fuc a1-3( Fuc a1-2( Gal a1-3) Gal b1-4) GIcNAc; this is referred to herein as a “terminal galactose”, this can (also) be bound by SJA; and

[0283] Fuc a1-2( Gal b1-3 GalNAc a1-3) Gal b1-4 GIcNAc; this is referred to herein as a “terminal galactose”, this can be bound by PNA.

[0284] Thus, items “terminal galactose residues” and “terminal N-acetylgalactosamine (GalNAc) residues” 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.As mentioned above, it is preferred that STL is used for binding internal N-acetyllactosamine (e.g. LacNAc). As such, any list of glycan markers outlined herein may optionally be absent “terminal N-acetylglucosamine (GIcNAc) residues”. Indeed any reference to “terminal N-acetylglucosamine (GIcNAc) residues” throughout this disclosure is totally optionally and can optionally be omitted from the associated aspect / embodiment. Although the term “terminal N-acetylglucosamine (GIcNAc) residues” may be optionally absent from a list of glycans, the list may continue to embrace “terminal galactose residues” and “internal N-acetyllactosamine residues” that may happen to comprise a GIcNAc component.

[0285] Noting lectins represent preferred binding partners, the skilled person understands which glycans said lectins bind to, and would thus understand the what the glycan is when described with reference to the lectin it is bound by. For example:

[0286] 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(1— >6) GIcNAc, a Fuc a(1— >2) GIcNAc, a Fuc a(1— >3) GIcNAc, and / or a Fuc a(1— >4) GIcNAc (preferably a Fuc a(1— >6) GIcNAc)” may be referred to as a terminal glycan residue capable of being bound by AAL;

[0287] terminal sialic acid that is an “alpha-2, 3-linked sialic acid” (preferably a terminal NeuAc a2-3 Gal b1-3 GalNAc residue) may be referred to as a terminal glycan residue capable of being bound by MAL-II;

[0288] terminal sialic acid that is an alpha-2, 6-linked sialic acid (preferably a terminal NeuAc a2-6 Gal b1-4 GIcNAc residue) may be referred to as a terminal glycan residue capable of being bound by SNA;

[0289] terminal N-acetylglucosamine (preferably a P(1 ,4)-linked GIcNAc) 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;

[0290] 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;

[0291] 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;

[0292] terminal galactose “Fuc a1-2 (Gal b1-3 GalNAc a1-3) Gal b1-4 GIcNAc” may be referred to as a terminal glycan residue capable of being bound by PNA;

[0293] terminal galactose “Fuc a1-3 (Fuc a1-2 (Gal a1-3) Gal b1-4) GIcNAc” may be referred to as a terminal glycan residue capable of being bound by SJA;terminal N-acetylgalactosamine (preferably GalNAc b1-4 GIcNAc b1-3 GalNAc b1-4 GIcNAc) may be referred to as a terminal glycan residue capable of being bound by SJA.

[0294] Plurality of glycan markers

[0295] Thus, in a suitable embodiment a method of the invention further comprises determining the presence of a plurality of glycan markers, and characterising the state of the particle on the basis of these determinations.

[0296] In a suitable embodiment the presence of three or more glycan markers is determined, or the presence of four or more glycan markers is determined, or the presence of five or more glycan markers is determined.

[0297] In a suitable embodiment presence of six or more glycan markers is determined, or the presence of seven or more glycan markers is determined, or the presence of eight or more glycan markers is determined, or the presence of nine or more glycan markers is determined, or the presence often or more glycan markers is determined.

[0298] In any such embodiment the plurality of glycan markers may each be selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; a N-acetylgalactosamine (GalNAc) residue; a fucose residue; a N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.

[0299] It is preferred that in any such embodiment the plurality of glycan markers may each be selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; a terminal N-acetylglucosamine (GIcNAc) residue; and an internal N-acetyllactosamine residue.

[0300] For the avoidance of doubt, any of the specific glycan markers mentioned above in respect of a ‘first glycan marker’, a ‘second marker’ or a ‘third or subsequent marker’ can likewise be used to further define the glycan markers in embodiments that discuss the determination of a ‘plurality of glycan marker’ or ‘three (and subsequent) or more glycan markers’; or indeed any embodiment that refers to a glycan marker.

[0301] In a suitable embodiment of a method of the invention, a determination is made in respect of twenty or more glycan markers. In a suitable embodiment of a method of the invention, adetermination is made in respect of 30 or more glycan markers. In a suitable embodiment of a method of the invention, a determination is made in respect of 40 or more glycan markers. In a suitable embodiment of a method of the invention, a determination is made in respect of 50 or more glycan markers. In a suitable embodiment of a method of the invention, a determination is made in respect of 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200 or more glycan markers.

[0302] In a suitable embodiment of a method of the invention, a determination is made using one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more binding partners capable of binding glycan markers. In a suitable embodiment of a method of the invention, a determination is using twenty or more binding partners capable of binding glycan markers. In a suitable embodiment of a method of the invention, a determination is made using 30 or more binding partners capable of binding glycan markers. In a suitable embodiment of a method of the invention, a determination is made using 40 or more binding partners capable of binding glycan markers. In a suitable embodiment of a method of the invention, a determination is made using 50 or more binding partners capable of binding glycan markers. In a suitable embodiment of a method of the invention, a determination is made using 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200 or more binding partners capable of binding glycan markers. Suitably the binding partners referred to are lectins. By way of example, the lectins may be human lectins. Alternatively, the lectins may be murine lectins.

[0303] In a suitable embodiment, one or more lectin used in a method of the invention, or provided in a kit of the invention, is a human lectin. In a suitable embodiment, each of the lectins used in a method of the invention, or provided in a kit of the invention, is a human lectin.

[0304] In a suitable embodiment, one or more lectin used in a method of the invention, or provided in a kit of the invention, is a murine lectin. In a suitable embodiment, each of the lectins used in a method of the invention, or provided in a kit of the invention, is a murine lectin.

[0305] Methods using non-glycan markers

[0306] In a method in accordance with the invention, the second marker may be a non-glycan marker. The use of a non-glycan marker may be particularly advantageous for determining certain states, such as the state as it relates to a stage of apoptosis. As will be discussed in theexamples section, combined use of a first glycan marker and a second non-glycan marker for phosphatidylserine can reveal apoptotic states that could not be resolved with prior art methods, including the emergence of pre-commitment (to apoptosis) state cells as early as 15 minutes after induction of apoptosis.

[0307] In such an example, suitably the second marker is selected from the group consisting of: a protein; a lipid; and a nucleic acid.

[0308] For example, a second marker may be a protein. In such an embodiment, the protein second marker may be selected from the group consisting of: a caspase (such as caspase-3); Bcl2; and Bcl2-associated X protein (BAX).

[0309] In a suitable embodiment, a second marker is a lipid. In such an embodiment, the lipid second marker may be phosphatidylserine (PS).

[0310] In a suitable embodiment of a method of the invention, the second marker is selected from the group consisting of: an apoptosis marker; a viability marker; and a cell cycle marker.

[0311] In such an example the apoptosis marker is selected from the group consisting of: a mid-stage apoptosis marker; and an apoptosis regulator protein. For example, a mid-stage apoptosis marker may be selected from the group consisting of: phosphatidylserine; and a caspase.

[0312] In a suitable embodiment of a method of the invention, presence of phosphatidylserine is determined by binding of a binding partner selected from the group consisting of: annexin V; and an antibody that binds to phosphatidylserine.

[0313] In a suitable embodiment of a method of the invention, an apoptosis regulator protein is selected from the group consisting of: Bcl2; and BAX.

[0314] In a suitable embodiment of a method of the invention, a viability marker is a nucleic acid. For example, the viability marker may be selected from the group consisting of: DNA; and RNA. In these embodiments, the presence of the viability marker may be determined by binding of a binding partner selected from the group consisting of: 7-AAD; eF780; SyTox Red; and SyTox Green.

[0315] Generally, in any suitable embodiment of a method of the invention, presence of the second, or subsequent, marker is determined by assessing binding of a binding partner for the marker.A binding partner, whether for a first, second, or subsequent marker, may suitably be labelled with a detection moiety.

[0316] In a suitable embodiment, the detection moiety is selected from the group consisting of: a fluorophore; a chromogen; a bead; a tag sequence; biotin; and an enzyme. In the case of a flurophore, this may be selected from the group consisting of: phycoerythrin (PE); and propidium iodide (PI).

[0317] Beads suitable for use as detection moieties in the methods or kits of the invention include magnetic beads, and beads (such gold, or other metal, beads) able to be detected via microscopy.

[0318] Suitable examples of enzymes that may be used as detection moieties in the methods or kits of the invention include horseradish peroxidase (which is also an example of a chromogen); and luciferase.

[0319] External and internal markers

[0320] In a suitable embodiment, one, more than one, or all of the markers may be endogenous markers.

[0321] In a suitable embodiment, one, more than one, or all of the markers may be exogenous markers.

[0322] In a method of the invention, the presence of a glycan first marker on a surface of a particle is determined. In a suitable embodiment of a method of the invention, the presence of the second, or subsequent, marker on the external surface of the particle is determined.

[0323] In a suitable embodiment of a method of the invention, the presence of the second, or subsequent, marker on an excreted particle (e.g. vesicle released from a cell) is determined.

[0324] In a suitable embodiment of a method of the invention, each marker is determined with respect to its presence on the external surface of the particle.

[0325] In a suitable embodiment of a method of the invention, the presence of the second, or subsequent, marker within the particle is determined.In a suitable embodiment of a method of the invention, the second, or subsequent, marker is the intracellular apoptosis marker mitochondrial membrane potential.

[0326] In a suitable embodiment of a method of the invention, the second, or subsequent, marker is an intracellular metabolite. A suitable intracellular metabolite may be selected from the group consisting of: free mannose; free pyruvate; ATP; reactive oxygen species; NADH; and NADPH.

[0327] In a suitable embodiment of a method of the invention, the presence of a second or subsequent marker may be determined with reference to an intracellular detection moiety (e.g. fluorescent detection moiety). Such an intracellular detection moiety may be a fluorescent protein. For example, a suitable fluorescent protein may be selected from the group consisting of: green fluorescent protein (GFP); blue green fluorescent protein (BFP); oryellow fluorescent protein (YFP).

[0328] A suitable second, or subsequent, marker may be an activity marker. In a suitable embodiment, an activity marker may be a detectable enzyme, such as luciferase.

[0329] While methods of the invention utilising binding partners and / or detection moieties to determine the presence of markers (whether the first, second, or subsequent markers) have been extensively considered above, the skilled person will recognise that the methods and kits of the invention need not be limited to such “indirect” approaches the detection of markers.

[0330] In a suitable embodiment of a method of the invention, the presence of a marker (whether the first, second, or subsequent marker) may be determined by a “direct” approach. Merely by way of example, mass spectroscopy may be used in such a direct approach to determining the presence of a marker.

[0331] The skilled person will be able to identify alternative approaches that may be used in the practice of the invention.

[0332] Methods of determination and characterisation

[0333] In a suitable embodiment of a method of the invention, the determination of the presence of the first marker comprises quantification of the first marker. In a suitable embodiment of a method of the invention, the determination of the presence of the second marker comprisesquantification of the second marker. In a suitable embodiment of a method of the invention, the determination of the presence of both the first and second markers comprises quantification of the first and second markers. In a suitable embodiment of a method of the invention, the determination of the presence of a third, or subsequent, marker comprises quantification of the third marker.

[0334] A suitable embodiment of a method of the invention comprises determination of the presence of each of the first, second, third, and subsequent markers comprises quantification of the markers.

[0335] In a suitable embodiment of a method of the invention, comprises comparing ratios of the markers.

[0336] In a suitable embodiment of a method of the invention, the state of a particle, or population of particles, is characterised by calculating a ratio of the first and second markers. Ratios including a third and subsequent markers, if present, may also be used in characterisation in such an embodiment.

[0337] The inventors have found that characterisation of particles, or populations of particles, on the basis of ratios of the first and second (and optionally third and subsequent) markers offers a number of advantages in practice that have not previously been appreciated. Dynamic changes between markers (some of which may be decreasing, while others are increasing) may enable characterisation on the basis of a calculated ratio to identify changes in state earlier than techniques based on “absolute” values of the same markers. Accordingly, methods of the invention in which particles, or populations of particles, are characterised on the basis of ratios of the markers determined may offer increased sensitivity as compared to previously known characterisation techniques.

[0338] In a suitable embodiment of a method of the invention, determination of the presence of the one or more, or all, of the markers is performed by array binding. In a suitable embodiment of a method of the invention, determination of the presence of the first and second markers is performed by array binding.

[0339] In a suitable embodiment, determination of the presence one or more, or all, of markers may be performed by flowing a sample comprising a particle described herein (or population of particles described herein) along a flow channel; applying a detection signal (e.g. light source such as a laser) for each of the first and second markers through the flow channel; anddetermining the presence of each assayed marker as an individual (single) particle passes through the detection signal.

[0340] In a suitable embodiment of a method of the invention, determination of the presence of the first and second markers is performed by flowing a sample comprising a particle described herein (or population of particles described herein) along a flow channel; applying a detection signal (e.g. light source such as a laser) for each of the first and second marker through the flow channel; and determining the presence of each assayed marker as an individual (single) particle passes through the channel.

[0341] Embodiments described in the paragraph above may be achieved by the use of flow cytometry (e.g. may be described as embodiments where marker determination is performed by flow cytometry).

[0342] In a suitable embodiment of a method of the invention, determination of the presence one or more, or all, of markers is performed by flow cytometry. In a suitable embodiment of a method of the invention, determination of the presence of the first and second markers is performed by flow cytometry. Advantageously, flow cytometry allows for the simultaneous determination of the presence of each assayed marker as an individual (single) particle passes through the channel.

[0343] Further advantages of flow cytometry include the ability to determine forward-scatter light (FSC) and / or side-scattered light for a particle described herein, which may also be determined fora particle in methods of the invention (e.g. determined simultaneously with the determination of a marker described herein). Thus, methods of the invention can additionally comprise determining a FSC-H, FSC-A, FSC-W, SSC-H, SSC-A, SSC-W for a particle (e.g. each individual assayed particle). Preferably, at least FSC-H and FSC-A (e.g. for each individual particle) are determined. As will be discussed elsewhere herein, these two latter parameters are notably advantageous in terms of moderating flow cytometry data.

[0344] A yet further advantage of flow cytometry includes the ability to perform particle sorting (e.g. Fluorescence-Activated Cell Sorting (FACS)), which may form an additional step of a method described herein, particularly in methods involving a population of particles (e.g. of heterogenous states), such that particles of a given (determined) state can be sorted, for example for further analysis such as genomics, transcriptomics, and / or proteomics.That being said, biophysical techniques (such as columns having a binding partner for a glycan of interest) can also be used to separate / sort particles of interest.

[0345] In a suitable embodiment of a method of the invention, determining the presence of one or more, or all, of the markers comprises determining the density of the marker, or markers, on the external surface of the particle (e.g. based on the surface area of the external surface).

[0346] In a suitable embodiment of a method of the invention, determining the presence of a glycan first marker comprises determining the density of the glycan first marker on the external surface of the particle (e.g. based on the surface area of the external surface).

[0347] In a suitable embodiment of a method of the invention, determining the presence of a second marker comprises determining the density of the second marker on the external surface of the particle (e.g. for external second markers).

[0348] In a suitable embodiment of a method of the invention, determining the presence of a third or subsequent marker comprises determining the density of the third or subsequent marker on the external surface of the particle (e.g. based on the surface area of the external surface).

[0349] In a suitable embodiment of a method of the invention, determining the presence of third or subsequent marker comprises determining the density of the third or subsequent marker on the external surface of the particle (e.g. for an external third or subsequent marker).

[0350] In a suitable embodiment of a method of the invention, determining the presence of one or more, or all, of the markers comprises determining the density of the second marker based on the volume of the particle. In a suitable embodiment of a method of the invention, determining the presence of a second marker comprises determining the density of the second marker based on the volume of the particle (e.g. for an intracellular second marker).

[0351] The ability to use a forward scatter signal to moderate the (raw) signal for a marker, to provide a density for a marker of the invention in / on a particle, demonstrates yet further advantages of flow cytometry analysis of first and second markers described herein. That being said, particle (e.g. cell) size and / or volume can also be interpreted by other means, including from an image (e.g. where the number of pixels of an image provide an indication of particle size and / or volume), such that the means used to interpret a particle’s size and / or volume, which can then be used to moderate a (raw) signal for a marker to provide a density for the marker in / on the particle, is not necessarily limited to embodiments that involve flow cytometry.In such methods determination of the presence of the first marker may be performed by flow cytometry, and determining the presence of a glycan first marker on the external surface of the particle further comprises measuring a level for said glycan first marker on the external surface of the particle; and

[0352] performing a moderation step comprising moderating the level for said glycan first marker using a level for a forward scatter signal (FSC level) for the particle.

[0353] Such a method may optionally comprise a step of determining the FSC level (e.g. for the same particle associated with the glycan first marker). In other words, a level for a marker (whether first, second, third or subsequent) can be determined for a given particle and subsequently moderated using an FSC determined for said same particle. The marker level is preferably determined as fluorescence intensity (e.g. from a fluorophore associated with a complementary binding partner bound to the marker), with said fluorescence intensity being preferably subjected to moderation (e.g. based on particle size / volume) as described herein that yields a value for density of the marker in / on a particle.

[0354] In a suitable embodiment of a method of the invention, the moderation step comprises dividing the level for the glycan first marker by the FSC (e.g. FSC-A or FSH-H, preferably FSC-A) e.g. to determine an indication of density of the first marker on the surface of the particle.

[0355] In a suitable embodiment of a method of the invention, determination of the presence of the second marker is performed by flow cytometry, and wherein determining the presence of the second marker comprises measuring a level for the second marker on the external surface of the particle; and

[0356] performing a moderation step comprising moderating the level for said second marker using a level for a forward scatter signal (FSC level) for the particle.

[0357] In such embodiments, the second marker is preferably also an external marker, for example the second marker may be a second glycan marker.

[0358] In a suitable embodiment of a method of the invention, determination of the presence of the third or subsequent marker is performed by flow cytometry, and wherein determining the presence of the third or subsequent marker comprises measuring a level for the second marker on the external surface of the particle; and

[0359] performing a moderation step comprising moderating the level for said third or subsequent marker using a level for a forward scatter signal (FSC level) for the particle.In such embodiments, the third or subsequent marker is preferably also an external marker, for example the third or subsequent marker may be a third glycan marker.

[0360] In a suitable embodiment of a method of the invention, the moderation step comprises dividing the level for the second (and / or third or subsequent marker where present) marker by the FSC (e.g. FSC-A or FSH-H, preferably FSC-A) level to determine an indication of density of the second marker on the surface of the particle. Again, said density (based on dividing by the FSC) may be said to be a “surface density”.

[0361] In a suitable embodiment of a method of the invention, determination of the presence of the second marker is performed by flow cytometry, and wherein determining the presence of the second marker comprises measuring a level for the second marker within the particle’s cell membrane (e.g. for a second marker that is an internal marker); and

[0362] performing a moderation step comprising moderating the level for said second marker using a level for a forward scatter signal (FSC) for the particle.

[0363] In a suitable embodiment of a method of the invention, determination of the presence of the third or subsequent marker is performed by flow cytometry, and wherein determining the presence of the third or subsequent marker comprises measuring a level for the second marker within the particle’s cell membrane (e.g. for a third or subsequent marker that is an internal marker); and

[0364] performing a moderation step comprising moderating the level for said second marker using a level for a forward scatter signal (FSC) for the particle.

[0365] In a suitable embodiment of a method of the invention, said moderation step comprises dividing the level for the first glycan marker by the FSC (e.g. FSC-A or FSH-H, preferably FSC-A) level to the power of 3 / 2 (FSC3 / 2) to determine an indication of volumetric density (e.g. vDen) of the first glycan marker within the particle.

[0366] In a suitable embodiment of a method of the invention, said moderation step comprises dividing the level for the second marker (and / or third or subsequent marker where present) by the FSC (e.g. FSC-A or FSH-H, preferably FSC-A) level to the power of 3 / 2 (FSC3 / 2) to determine an indication of volumetric density (e.g. vDen) of the second marker within the particle. In other words, said moderation step may comprise dividing the level for a marker described herein by FSC3 / 2to determine an indication of volumetric density of the marker within the (e.g. one or more) particle(s).Thus, as already mentioned elsewhere herein, an FSC value for an individual (e.g. each) particle can be determined, together with a determination for the marker (first, second; and third or subsequent where present) for said same individual particle.

[0367] It is preferred that a level for a marker (whether first, second, third or subsequent) can be determined for a given particle and subsequently moderated using an FSC determined for said same particle. That being said, methods of the invention may additionally (or alternatively) involve a moderation step that comprises determining an average indication (e.g. level) of a marker (whether first, second, third or subsequent) present for a plurality of particles; determining an average forward scatter signal for said plurality of particles; and dividing the average indication (e.g. level) of the marker present for said plurality of particles based on the average forward scatter signal for said plurality of particles.

[0368] It is preferred that any determination for an external marker level (such as an external glycan, or external lipid such as phosphatidyl serine optionally determined with annexin A) involves moderation by surface density as described herein. In addition, it is preferred that an internal marker (e.g. 7-AAD) (e.g. its level) is moderated by volumetric density as described herein. It is preferred that all marker levels determined are moderated appropriately, thus moderation based on surface density is preferably coupled with moderation for volumetric density where first and second markers are external and internal, respectively. Where third or subsequent markers are involved, it is likewise preferred that their level(s) are moderated based on surface density for an external third (or subsequent) marker, else based on volumetric density for an internal third (or subsequent) marker.

[0369] Although it is preferred that an FSC (as used for a moderation step described herein) derives from the assayed particle, a moderation step can alternatively (or additionally) may involve a FSC for a control particle / sample, such as a bead of known size. For example, an FSC for a particle of known size may be provided, e.g. wherein size calibration data has been determined which associates FSC signals with particle size based on obtained flow cytometer data for the control particle; and wherein moderating a level for a marker (e.g. first, second, third or subsequent) for an assayed particle based on an indication of size for said (assayed) particle uses the forward scatter signal and the size calibration data.

[0370] These levels forthe control data may be accessed (e.g. from data from a control flow cytometry step previously conducted), or may be generated as a step of a method described herein. For example, a method of the invention may comprise obtaining flow cytometer data for a controlsample, wherein the control sample comprises one or more particles of known size, and wherein the flow cytometer data comprises a forward scatter signal, FSC, for each one or more particles within the control sample; and determining size calibration data which associates FSC signals with particle size, based on the obtained flow cytometer data for the control sample; wherein moderating the level of the marker present for an (assayed) particle (e.g. within a test sample) based on an indication of size for said (assayed) particle uses the forward scatter signal and the size calibration data.

[0371] In such embodiments, moderating the level for the marker present for the (assayed) particle (e.g. within the test sample) may further comprise: determining an indication of size for said (assayed) particle using the forward scatter signal for said (assayed) particle and the size calibration data; and dividing the level of the marker present based on the indication of size to determine an indication of density of the marker for the (assayed) particle; optionally wherein dividing the level of the marker present based on the indication of size comprises: dividing the level for the marker present by the indication of size to determine an indication of surface density of the marker for the (assayed) particle (e.g. for an external marker); and / or dividing the level of the marker present by the indication of size to the power of3 / 2, (the indication of size)372, to determine an indication of volumetric density of the marker within the particle (e.g. for an internal marker).

[0372] The skilled person understands that a method of the invention may involve performing a moderation step as described herein, and graphically plotting the moderated level(s) of the marker present for the (assayed) particle(s), e.g. as part of a computer-implemented method step for graphical analysis of flow cytometry data. For example, by graphically plotting a moderated level for the first marker on a first axis, and plotting a moderated level for the second marker on a second axis.

[0373] For methods that involve a population of particles, methods of the invention may comprise obtaining flow cytometer data for a population of particles (e.g. within a biological sample), wherein the flow cytometer data comprises: (i) a first level of a first glycan marker present for each particle within the population of particles; (ii) a second level for a second marker present for each particle within the population of particles; and obtaining an indication of size for each of the particles within the population; moderating the first indication of the first marker present for each particle using the indication of size for said particle; moderating the second level of the second marker present for each particle, using indication of size (or volume) for said particle; graphically plotting the moderated level of the first marker on a first axis, and themoderated level of the second marker on a second axis; and identifying a first subpopulation of particles within the population of particles based on the graphical plot.

[0374] Methods of the invention may involve charactering the state of the particle (or a population or particles) as being dead (or apoptotic or necrotic), the method comprising: obtaining flow cytometer data for a particle (or a population of particles) e.g. within a sample, wherein the particle (or population of particles) has / have been marked with a viability marker, such that the flow cytometer data comprises an indication of the viability marker present for the particle (or each particle within the population of particles); obtaining an indication of size for the particle (or each of the particles); moderating the indication (e.g. level such as fluorescence level) of the viability marker present for the particle (or each particle) using the indication of size for said particle (or each particle); identifying state for the particle (or population or subpopulation of particles) based on the corrected indication of the viability marker, wherein the state for the particle (or population or subpopulation of particles) is characterised as being dead (or apoptotic or necrotic).

[0375] For completeness, although a determination of a marker (first, second, third etc) herein is preferably performed by flow cytometry, a level for a marker (as determined by flow cytometry) may also make use of information other than the FSC fora particle. For example, the indication of size may be obtained using image analysis of at least one image of an (assayed) particle. For example, the flow cytometer data for a test sample (having a particle being assayed by the invention) may comprise imaging flow cytometer data, comprising at least one image of the particle(s) within the sample, wherein obtaining the indication of size for the particle(s) within the sample comprises determining cell size based on image analysis of the at least one image.

[0376] Details of the particles to be investigated

[0377] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is from a prokaryotic source. Suitably in such an embodiment of a method of the invention, the particle comprising a cell membrane is from a bacterial source.

[0378] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is from a eukaryotic source. Suitably in an embodiment of such a method of the invention, the particle comprising a cell membrane is from a plant source. Suitably in an embodiment of such a method of the invention, the particle comprising a cell membrane is from an animal source.A suitable particle may be from a mammalian source, such as a human source. A suitable particle may be from an experimental animal source. A suitable particle may be from a murine source.

[0379] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is from a wild-type source.

[0380] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is from a genetically modified source.

[0381] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is selected from the group consisting of: a cell; a cell component (such as a vesicle); and a free cell membrane (such as a ghost). The particle may be a vesicle that has derived from a cell in the same test sample being assayed in a method of the invention.

[0382] Except for where context requires otherwise, references to “cells” in the context of the present invention should also be taken as referring to components of cells (e.g. vesicles, organelles, cell apparatus, or cell fragments) derived from cells (e.g. from a cell in the same test sample being assayed in a method of the invention), and vice versa. Similarly, references in the specification to “particles” should be taken as appliable to cells and their components, unless context requires otherwise.

[0383] It will be appreciated that suitable cells may be found in a wider range of samples, including (but not limited to) tissue samples, blood samples, sputum samples, semen samples, and samples from tissue lavage.

[0384] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is a cell. Suitably the cell is an uncompromised cell. Suitable the cell is a healthy cell. Suitably the cell is a pre-apoptotic cell.

[0385] A pre-apoptotic cell may be a cell that is pre-committed to apoptosis, such as a cell that has been exposed to conditions (such as staurosporine) that induce apoptosis, but that does not yet exhibit caspase 3 activation. A pre-apoptotic cell may be a cell that is committed to apoptosis, such as a cell that has been exposed to conditions (such as staurosporine) that induce apoptosis, and in which caspase 3 activation is detectable.In a suitable embodiment of a method of the invention, the cell is an apoptotic cell.

[0386] In a suitable embodiment of a method of the invention, cell is a necrotic cell.

[0387] In a suitable embodiment, a method in accordance with the invention may be practiced in respect of a particle, or population of particles, in particular cells or vesicles, representative of one or more condition from the group consisting of: a cell undergoing autophagy; a quiescent cell (for example a cell at GO of the cell cycle); an actively cycling cell (such as a cell at G1 , S, G2 or M of the cell cycle); an oxidatively stressed cell; a glucose deprived cell; a metabolically compromised cell; and an infected cell (for example, infected with a bacterial infection, an intracellular bacterial infection, a viral infection, or a fungal infection).

[0388] In a suitable embodiment of a method of the invention, the cell is selected from the group consisting of: a blood cell; a stem cell; a neuronal cell; an immune cell; an epithelial cell; an endothelial cell (such as a HUVEC); a bone cell; a muscle cell; a fat cell; a sex cell; a cancer cell; a liver cell; a fibroblast; a cell of an established cell line; and an immortalised cell.

[0389] A suitable blood cell may be selected from the group consisting of: a lymphocyte (such as a T cell or B cell); an erythrocyte; a macrophage; a monocyte; and a granulocyte (such as a neutrophil or eosinophil).

[0390] A suitable stem cell may be selected from the group consisting of: a mesenchymal stem cell; a pluripotent stem cell (such as an iPSC); preferably a mesenchymal stem cell.

[0391] In a suitable embodiment of a method of the invention, the cell is a fusion cell or multinucleate cell.

[0392] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is a cell component (optionally derived from a cell in the same test sample being assayed in a method of the invention).

[0393] In a suitable embodiment, the cell component is selected from the group consisting of: an organelle (for example, a nucleus, fused nucleus, nuclear pore, lysosome, endosome, mitochondrion, chloroplast); a fused organelle (for example, a phagolysosome); cell apparatus (for example, an endoplasmic reticulum or Golgi body); and a cell fragment (for example, an apoptotic fragment, such as one containing broken parts of DNA).In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is a vesicle.

[0394] Suitably, a vesicle is derived from an uncompromised cell. Suitably, a vesicle is derived from a healthy cell. Suitably, a vesicle is derived from a pre-apoptotic cell. Suitably, a vesicle is derived from an apoptotic cell. Suitably, a vesicle is derived from a necrotic cell.

[0395] In a suitable embodiment of a method of the invention, the particle is from an animal.

[0396] In a suitable embodiment of a method of the invention, the particle comprising a cell membrane is live. For example, it may be said that the particle (at the time of determining a marker described herein) retains its inherent ability to express a change of state that occurs along a biological pathway. The particle may be a particle (or a progeny particle thereof such as a vesicle) predisposed to undergo such a biological pathway. The particle may be a particle (or a progeny particle thereof such as a vesicle) that has been induced to undergo such a biological pathway. Thus, methods of the invention can allow for probing a dynamic (or realtime) biological pathway, while it is still happening.

[0397] It is preferred that methods of the invention involve analysis of a dynamic particle / particle populations, the assayed particle / particle populations retaining the propensity to undergo state change (e.g. instead of being fixed).

[0398] Suitably, the particle has not been subjected to a lysis, permeabilization and / or fixation step. Suitably, the particle comprising a cell membrane is not lysed. Suitably, the particle comprising a cell membrane is not permeabilised. Suitably, the particle comprising a cell membrane is not fixed.

[0399] Thus, in a preferable embodiment, the particle(s) (e.g. test sample comprising the same) has / have 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 particle(s) (e.g. test sample comprising the same) has / have 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 particle(s) has not been subjected to any of such cell lysis / permeabilization / acid treatment steps).

[0400] Suitably, the cell membrane of the particle is intact. For example, it may be said that a particle (or at least one or a subset of particles) described here comprises a cell membrane thatencloses (e.g. continues to enclose) liquid and / or cytoplasm, such that the cell membrane retains the lipid bi-layer structure and semi-permeable properties indicative of a living cell (or vesicle derived therefrom). In a suitable embodiment, a method of the invention is practiced using a sample comprising, or consisting essentially of, intact cells.

[0401] It may be said that the glycan is present as an integral part of the outer membrane of the assayed particle. The glycan may be bound directly or indirectly to the outer membrane. The glycan may, for example, be bound indirectly via a cell surface protein or lipid (e.g. glycoprotein / glycolipid).

[0402] Suitably, the particles have not been subjected to a pre-enrichment step for specific cell state (e.g. specific state of a biological process within a given same cell type). Methods of the invention may be performed with a heterogenous population of particles of different states (e.g. particles not having been enriched to a provide a homogenous population of particle state).

[0403] Non-agnostic methods

[0404] Apoptosis

[0405] In a suitable embodiment of a method of the invention, the particle is putatively apoptotic, and the second marker comprises an apoptosis marker. A suitable apoptosis marker may be as discussed elsewhere in the specification.

[0406] In a suitable embodiment of such a method of the invention, the particle is characterised as being:

[0407] • Non-apoptotic;

[0408] • Pre-committed to apoptosis (e.g. pre-commitment state);

[0409] • Committed to apoptosis (e.g. commitment state); or

[0410] • Post-committed to apoptosis (e.g. post-commitment state).

[0411] That being said, the inventors have demonstrated that a more particular substates (e.g. a plurality of “pre-committed” states) can be identified by methods of the invention, particularly based on differences in the first glycan marker amongst such substates.In the embodiments below, the first and second (preferably first to third) pre-commitment state cells are preferably associated with a determined ‘absence’ for a second marker that is phosphatidylserine (e.g. annexin A) e.g. a level that is not (significantly) higher that a non-apoptotic, or health, cell. For at least second pre-commitment state cell, and subsequent third to fifth and eighth commitment or post-commitment state particles, said particles are preferably associated with a determined ‘presence’ for a second marker that is phosphatidylserine (e.g. annexin A); preferably wherein the level of phosphatidylserine progressively increases, e.g. from a lowest level in a first commitment or post-commitment state particle, to a highest level in a sixth commitment (or post-commitment) state particle. The sixth and seventh commitment (or post commitment) state particles are preferably associated with a determined ‘absence’ for a second marker that is phosphatidylserine (e.g. annexin A).

[0412] A particle may be characterised as a first pre-commitment (to apoptosis) state cell [e.g. about 15 to 30 minutes, or 3%-15%, through the pre-commitment to apoptosis]. For the characterisation of such first pre-commitment state cell, the first glycan marker is preferably a LacNac (e.g. at the -1 position) or a terminal fucose. Such LacNac may be a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 bonds. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1 ,4) GIcNAc. Optionally, a binding partner for a first pre-commitment state cell may be STL and / or AAL.

[0413] A particle may be characterised as a second pre-commitment state cell (e.g. M1h pop) that derives from (or represents a further progression along the biological pathway of) said first pre-commitment state cell. For the characterisation of such second pre-commitment state cell, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc; Fuc (a1 ,2) GIcNAc; Fuc (a1 ,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a second precommitment state cell may be AAL, GNA, or SNA.

[0414] A particle may be characterised as a third pre-commitment state cell (e.g. M7h pop) that derives from (or represents a further progression along the biological pathway of) said second pre-commitment state cell. For the characterisation of such third pre-commitment state cell, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc; Fuc (a1 ,2) GIcNAc; Fuc (a1 ,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialicacid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a third precommitment state cell may be AAL, GNA, or SNA.

[0415] A particle may be characterised as a first commitment (to apoptosis) state cell (e.g. M7’ pop) that derives from (or represents a further progression along the biological pathway of) said third pre-commitment state cell. For the characterisation of such first commitment state cell, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc; Fuc (a1 ,2) GIcNAc; Fuc (a1 ,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a first commitment state cell may be AAL, GNA, or SNA.

[0416] A particle may be characterised as a second commitment state cell (e.g. M” pop) that derives from (or represents a further progression along the biological pathway of) said first commitment state cell. For the characterisation of such second commitment state cell, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1 ,4) GIcNAc. Such terminal mannose may be an alpha-1 , 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a second commitment state cell may be AAL, GNA, or SNA.

[0417] A particle may be characterised as a third commitment (to apoptosis) state particle (e.g. Pop 4) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said second commitment state cell. For the characterisation of such third commitment state cell, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a third commitment state cell may be AAL, GNA, or SNA.

[0418] A particle may be characterised as a fourth commitment state particle (e.g. Pop 5) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said third commitment state particle. For the characterisation of such fifth commitment state particle, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc;Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a fourth commitment state particle may be AAL, GNA, or SNA

[0419] A particle may be characterised as a fifth commitment state particle (e.g. Pop 6) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle. For the characterisation of such fifth commitment state particle, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1, 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a fifth commitment state particle may be AAL, GNA, or SNA.

[0420] A particle may be characterised as a sixth commitment or post-commitment state particle [e.g. Pop 7] that presents as a necrotic body state particle that derives from (or represents a further progression along the biological pathway of) said fifth commitment state particle (e.g. Pop 5). For the characterisation of such sixth commitment or post-commitment state particle, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1 ,4) GIcNAc. Such terminal mannose may be an alpha-1 , 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a sixth commitment or post-commitment state particle may be AAL, GNA, or SNA.

[0421] A particle may be characterised as a seventh commitment or post-commitment state particle [e.g. Pop 8] presenting as a necrotic body state particle that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle (e.g. Pop 5). For the characterisation of such seventh commitment or post-commitment state particle, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1 ,6) GIcNAc; Fuc (a1 ,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1,4) GIcNAc. Such terminal mannose may be an alpha-1 , 3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a seventh commitment or post-commitment state particle may be AAL, GNA, or SNA.

[0422] A particle may be characterised as an eighth commitment or post-commitment state particle that presents as a vesicle [e.g. Pop 9] that derives from (or represents a further progressionalong the biological pathway of) said fifth commitment state particle (e.g. which is Pop 6). For the characterisation of such eighth commitment or post-commitment state particle, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1,3) GIcNAc; or Fuc (a1 ,4) GIcNAc. Such terminal mannose may be an alpha-1 ,3-linked mannose (e.g. alpha-1 ,3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for an eighth commitment or post-commitment state particle may be AAL, GNA, or SNA.

[0423] A particle may be characterised as a vesicle state particle [e.g. Pop 10] that derives from blebs of healthy cells. For the characterisation of such vesicle state particle that derives from blebs of healthy cells, the first glycan marker is preferably a terminal fucose, a terminal mannose or a terminal sialic acid. Such terminal fucose may be Fuc (a1,6) GIcNAc; Fuc (a1,2) GIcNAc; Fuc (a1 ,3) GIcNAc; or Fuc (a1 ,4) GIcNAc. Such terminal mannose may be an alpha-1 ,3-linked mannose (e.g. alpha-1, 3-linked Man3GlcNAc2). Such terminal sialic acid may be an alpha-2, 6-linked sialic acid (e.g. NeuAc a2-6 Gal b1-4 GIcNAc). Optionally, a binding partner for a vesicle state particle that derives from blebs of healthy cells may be AAL, GNA, or SNA. Determining the state of apoptosis may benefit from determining a level for more than one glycan.

[0424] In a suitable embodiment the invention provides a method comprising

[0425] i. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three) glycan markers on the external surface of the particle, ii. comparing each level determined in step (i) with that of a corresponding reference value indicative of non-apoptotic (e.g. healthy) cells to determine a level change for each of the (e.g. two) glycan markers; and

[0426] Hi. characterising the state of the particle on the basis of these comparisons e.g. wherein the state is indicative of a stage of an apoptosis pathway to which the particle belongs.

[0427] Suitably, in such an embodiment of the invention, said at least one (preferably at least two, more preferably at least three) glycan markers are selected from the list consisting of a terminal fucose residue, a terminal mannose residue, and a terminal sialic acid residue; and wherein the particle is characterised as:

[0428] as a first pre-commitment (to apoptosis) state cell [e.g. about 15 to 30 minutes, or 3%- 10%, through the pre-commitment to apoptosis] when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease for internal LacNAc (e.g. a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 bonds, or an internal LacNAc bindable by STL), and a decrease for terminal fucose (e.g. bindable byAAL); a second pre-commitment state cell that derives from (or represents a further progression along the biological pathway of) said first pre-commitment state cell (e.g. M1 h pop), when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0429] o a decrease (e.g. about 1.7 fold) for terminal fucose, a decrease (e.g. about 1.9 fold) for terminal mannose, and a decrease (e.g. about 3.1 fold) for terminal sialic acid;

[0430] a third pre-commitment state cell that derives from (or represents a further progression along the biological pathway of) said second pre-commitment state cell, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0431] o a decrease (e.g. about 2.2 fold) for terminal fucose, a decrease (e.g. about 1.4 fold) for terminal mannose, and a decrease (e.g. about 2.0 fold) for terminal sialic acid;

[0432] a first commitment state cell that derives from (or represents a further progression along the biological pathway of) said third pre-commitment state cell, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0433] o a decrease (e.g. about 2 fold) for terminal fucose, a decrease (e.g. about 1.1 fold) for terminal mannose, and a decrease (e.g. about 1.6 fold) for terminal sialic acid;

[0434] a second commitment state cell that derives from (or represents a further progression along the biological pathway of) said first commitment state cell (e.g. M” pop) , when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0435] o a decrease (e.g. about 1.4 fold) for terminal fucose, an increase (e.g. about 1.2 fold) for terminal mannose, and substantially no level change for terminal sialic acid;

[0436] a third commitment (to apoptosis) state particle (e.g. Pop 4) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said second commitment state cell, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 7.4 fold) for terminal fucose, a decrease (e.g. about 4.7 fold) for terminal mannose, and a decrease (e.g. about 5.7 fold) for terminal sialic acid;

[0437] a fourth commitment state particle (e.g. Pop 5) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said third commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined: o a decrease (e.g. about 5.0 fold) for terminal fucose, a decrease (e.g. about 2.5 fold) for terminal mannose, and a decrease (e.g. about 3.9 fold) for terminal sialic acid;

[0438] a fifth commitment state particle (e.g. Pop 6) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0439] o a decrease (e.g. about 1.1 fold) for terminal fucose, an increase (e.g. about 2.5 fold) for terminal mannose, and an increase (e.g. about 8.4 fold) for terminal sialic acid;

[0440] a sixth commitment or post-commitment state particle that presents as a necrotic body state particle [e.g. Pop 7] that derives from (or represents a further progression along the biological pathway of) said fifth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0441] o a decrease (e.g. about 1.3 fold) for terminal fucose, an increase (e.g. about 2.0 fold) for terminal mannose, and a decrease (e.g. about 51.6 fold) for terminal sialic acid;

[0442] a seventh commitment or post-commitment state particle presenting as a necrotic body state particle [e.g. Pop 8] that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0443] o a decrease (e.g. about 4.9 fold) for terminal fucose, a decrease (e.g. about 4.1 fold) for terminal mannose, and a decrease (e.g. about 10.8 fold) for terminal sialic acid;

[0444] an eighth commitment or post-commitment state particle that presents as a vesicle [e.g. Pop 9] that derives from (or represents a further progression along the biological pathway of) said fifth commitment state particle when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.5 fold) for terminal fucose, an increase (e.g. about 2.9 fold) for terminal mannose, and an increase (e.g. about 8.3 fold) for terminal sialic acid; or

[0445] a vesicle state particle [e.g. Pop 10] that derives from blebs of healthy cells (e.g. M pop), when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:

[0446] o a decrease (e.g. about 1.5 fold) for terminal fucose, an increase (e.g. about 3.1 fold) for terminal mannose, and an increase (e.g. about 9.4 fold) for terminal sialic acid.

[0447] Suitably, in an embodiment of such an invention described in the preceding paragraphs: the terminal fucose is bindable by AAL (or wherein the terminal fucose is Fuc (a1,6) GIcNAc);

[0448] the terminal mannose is bindable by GNA (or wherein the terminal mannose is an alpha-1, 3-linked mannose, such as alpha-1, 3-linked Man3GlcNAc2); and

[0449] the terminal sialic acid is bindable by SNA (or wherein the terminal sialic acid is an alpha-2, 6-linked sialic acid, such as NeuAc a2-6 Gal b1-4 GIcNAc).

[0450] In any such embodiment (for example as set out in the preceding paragraphs) the cell may be a cell 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).

[0451] Suitably, in such embodiments, the cell is a stem cell; preferably a mesenchymal stem cell; more preferably a bone marrow mesenchymal stem cell.

[0452] Cell cycle

[0453] A particle may be characterised as being a cell in a particular stage of the cell cycle (e.g. where each cell cycle represents a ‘state’), for example a particle may be characterised as an S1 phase cell, a mid-S phase cell, an S2 phase cell, a G2 / M checkpoint phase cell, ora G1 phase cell. For such embodiments, the first glycan marker may be a terminal sialic residue, a terminal galactose, a terminal fucose, a terminal mannose, internal LacNAc residue (e.g. at the -1 position), or a terminal GIcNac residue described herein.

[0454] In a suitable embodiment of a method of the invention comprises:i. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers on the external surface of the particle,

[0455] ii. comparing each level determined in step (i) with that of a corresponding reference value (preferably density) indicative of CD4-CD8- lymphoid cells (e.g. B cells) to determine a level (preferably density) change for each of the (e.g. at least two) glycan markers; and

[0456] Hi. characterising the state of the particle on the basis of these comparisons e.g. wherein the state is indicative of a stage of a T-cell differentiation pathway to which the particle belongs.

[0457] In a suitable embodiment of such a method of the invention, said at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers are selected from the list consisting of (i) a terminal N-Acetylglucosamine residue bindable by GSIMP (“GSIMP residue”), a terminal sialic residue bindable by MAL-II (“MAL-II residue”), a terminal sialic residue bindable by SNA (“SNA residue”), a terminal galactose bindable by PNA (“PNA residue”), a terminal fucose bindable by SJA (“SJA residue”), a terminal mannose bindable by NPL (“NPL residue”), a terminal galactose or mannose residue bindable by ConA (“ConA residue”), a terminal fucose bindable by AAL (“AAL residue”), an internal LacNAc residue bindable by STL (“STL residue”); and wherein the particle is characterised as:

[0458] a CD4-CD8+ T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0459] o a decrease (e.g. about 1.4 fold) for the “GSIMP” residue, an increase (e.g.

[0460] about 2.8 fold) for the “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, an increase (e.g. about 1.1 fold) for the “PNA residue”, a decrease (e.g. about 1.3 fold) for the “SJA residue”, a decrease (e.g. about 1.5 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “NPL residue”, a decrease (e.g. about 1.1 fold) for the “ConA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, and a decrease (e.g. about 1.5 fold) for the “STL residue”;

[0461] a CD8+(Dim)CD4- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0462] o a decrease (e.g. about 1.1 fold) for the “GSIMP” residue, an increase (e.g.

[0463] about 2.4 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, adecrease (e.g. about 1.3 fold) for the “SJA residue”, an increase (e.g. about 1.3 fold) for the “GNA residue”, an increase (e.g. about 1.4 fold) for the “NPL residue”, an increase (e.g. about 1.1 fold) for the “ConA residue”, an increase (e.g. about 1.3 fold) for the “AAL residue”, and an increase (e.g. about 1.4 fold) for the “STL residue”;

[0464] a CD8+(High)CD4- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0465] o a decrease (e.g. about 1.6 fold) for the “GSIMP” residue, an increase (e.g.

[0466] about 2.9 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, an increase (e.g. about 1.2 fold) for the “PNA residue”, a decrease (e.g. about 1.3 fold) for the “SJA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.5 fold) for the “NPL residue”, a decrease (e.g. about 1.2 fold) for the “ConA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, and a decrease (e.g. about 1.7 fold) for the “STL residue”;

[0467] a CD4+CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0468] o a decrease (e.g. about 3.3 fold) for the “GSIMP” residue, an increase (e.g.

[0469] about 2.6 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.0 fold) for the “PNA residue”, a decrease (e.g. about 1.2 fold) for the “SJA residue”, a decrease (e.g. about 3.3 fold) for the “GNA residue”, a decrease (e.g. about 3.2 fold) for the “NPL residue”, a decrease (e.g. about 1.4 fold) for the “ConA residue”, a decrease (e.g. about 1.5 fold) for the “AAL residue”, and a decrease (e.g. about 6.5 fold) for the “STL residue”;

[0470] a CD4+(Dim)CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0471] o an increase (e.g. about 1.3 fold) for the “GSIMP” residue, an increase (e.g.

[0472] about 1.7 fold) for the “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, an increase (e.g. about 9.7 fold) for the “PNA residue”, a decrease (e.g. about 3.1 fold) for the “SJA residue”, an increase (e.g. about 1.5 fold) for the “GNA residue”, an increase (e.g. about 1.8 fold) for the “NPL residue”, an increase (e.g. about 1.3 fold) for the “ConA residue”, an increase(e.g. about 1.6 fold) for the “AAL residue”, and an increase (e.g. about 4.8 fold) for the “STL residue”; or

[0473] a CD4+(High)CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0474] o a decrease (e.g. about 3.0 fold) for the “GSIMP” residue, an increase (e.g.

[0475] about 2.6 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 4.6 fold) for the “PNA residue”, a decrease (e.g. about 1.2 fold) for the “SJA residue”, a decrease (e.g. about 4.1 fold) for the “GNA residue”, a decrease (e.g. about 3.9 fold) for the “NPL residue”, a decrease (e.g. about 1.5 fold) for the “ConA residue”, an decrease (e.g. about 1.5 fold) for the “AAL residue”, and a decrease (e.g. about 7.5 fold) for the “STL residue”.

[0476] In a suitable example, an embodiment of a method of the invention comprises

[0477] i. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers on the external surface of the particle,

[0478] ii. comparing each level determined in step (i) with that of a corresponding reference value (preferably density) indicative of G1 phase cell to determine a level (preferably density) change for each of the (e.g. at least two) glycan markers; and

[0479] Hi. characterising the state of the particle on the basis of these comparisons e.g wherein the state is indicative of the cell cycle stage to which the particle belongs.

[0480] In such method, said at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers may be selected from the list consisting of a terminal sialic residue bindable by MAL-II (“MAL-II residue”), a terminal sialic residue bindable by SNA (“SNA residue”), a terminal mannose bindable by GNA (“GNA residue”), a terminal fucose bindable by AAL (“AAL residue”), an internal LacNAc residue bindable by STL (“STL residue”), a terminal galactose bindable by PNA (“PNA residue”), a terminal galactose or terminal GalNAc bindable by SJA (“SJA residue”), a terminal mannose bindable by NPL (“NPL residue”);

[0481] wherein the particle is a CD8+(Dim) T cell (e.g. undergoing division) that has been activated by cross-linking CD3 and CD28, and the corresponding reference value (preferably density) is indicative of the first G1 phase of the CD8+(Dim) T cell subsequent to activation;

[0482] and wherein the particle is characterised as:an S1 phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0483] o a decrease (e.g. about 1.7 fold) forthe “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 1.2 fold) for the “GNA residue”, an increase (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 2.7 fold) forthe “STL residue”, an increase (e.g. about 1.1 fold) for the “PNA residue”, and an increase (e.g. about 1.3 fold) forthe “SJA residue”, a mid-S phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0484] o a decrease (e.g. about 1.9 fold) forthe “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.4 fold) for the “GNA residue”, substantially no level change forthe “AAL residue”, a decrease (e.g. about 3.1 fold) for the “STL residue”, a decrease (e.g. about 1.2 fold) for the “PNA residue”, and substantially no change forthe “SJA residue”;

[0485] an S2 phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0486] o a decrease (e.g. about 1.8 fold) forthe “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.3 fold) forthe “STL residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, and a decrease (e.g. about 1.8 fold) for the “SJA residue”, a G2M S phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0487] o a decrease (e.g. about 1.4 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 1.5 fold) for the “GNA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 2.7 fold) forthe “STL residue”, a decrease (e.g. about 1.5 fold) for the “PNA residue”, and substantially no change for the “SJA residue”;

[0488] an G1 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0489] o a decrease (e.g. about 1.7 fold) forthe “MAL-II” residue, an increase (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.3 fold) for the “GNAresidue”, substantially no level change for the “AAL residue”, a decrease (e.g. about 2.8 fold) for the “STL residue”, a decrease (e.g. about 1.2 fold) for the “PNA residue”, and an increase (e.g. about 1.5 fold) for the “SJA residue”, an S1 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0490] o a decrease (e.g. about 2 fold) for the “MAL-II” residue, an increase (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.4 fold) for the “GNA residue”, substantially no level change for the “AAL residue”, a decrease (e.g. about 3.3 fold) for the “STL residue”, a decrease (e.g. about 1.4 fold) for the “PNA residue”, and a decrease (e.g. about 1.2 fold) for the “SJA residue”, a mid-S phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0491] o a decrease (e.g. about 2.3 fold) for the “MAL-II” residue, a decrease (about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.8 fold) for the “STL residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, and a decrease (e.g. about 1.6 fold) for the “SJA residue”, an S2 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0492] o a decrease (e.g. about 2.7 fold) for the “MAL-II” residue, a decrease (about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.3 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.1 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 2.9 fold) for the “SJA residue”, a G2M S phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0493] o a decrease (e.g. about 2.3 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.2 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 1.7 fold) for the “SJA residue”;a G1 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0494] o a decrease (e.g. about 1.8 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.6 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.8 fold) for the “STL residue”, a decrease (e.g. about 1.4 fold) for the “PNA residue”, and a decrease (e.g. about 1.3 fold) for the “SJA residue”, an S1 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0495] o a decrease (e.g. about 2.0 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 4.0 fold) for the “STL residue”, a decrease (e.g. about 1.5 fold) for the “PNA residue”, and a decrease (e.g. about 1.5 fold) for the “SJA residue”, a mid-S phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0496] o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.1 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.4 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 2.3 fold) for the “SJA residue”, an S2 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0497] o a decrease (e.g. about 2.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.6 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 4.8 fold) for the “SJA residue”, a G2M S phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0498] o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.2 fold) for the “GNAresidue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”; an G1 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0499] o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 2.5 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.7 fold) for the “STL residue”, a decrease (e.g. about 1.9 fold) for the “PNA residue”, and a decrease (e.g. about 2.6 fold) for the “SJA residue”, an S1 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0500] o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 2.3 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 5.0 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 3.2 fold) for the “SJA residue”, a mid-S phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0501] o a decrease (e.g. about 2.0 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.7 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.4 fold) for the “STL residue”, a decrease (e.g. about 2.7 fold) for the “PNA residue”, and a decrease (e.g. about 3.8 fold) for the “SJA residue”, an S2 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0502] o a decrease (e.g. about 2.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.6 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 4.8 fold) for the “SJA residue”,a G2M S phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0503] o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.2 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”; a G1 phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0504] o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 3.2 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 10.6 fold) for the “SJA residue”; an S1 phase (fifth division post-activation) CD8+(Dim) T cell, when at least two (preferably at least three, more preferably at least four) of the following level changes are determined:

[0505] o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 2.8 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 3.0 fold) for the “PNA residue”, and a decrease (e.g. about 4.1 fold) for the “SJA residue”; a mid-S phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0506] o a decrease (e.g. about 1.9 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 3.1 fold) for the “GNA residue”, a decrease (e.g. about 1.5 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 3.3 fold) for the “PNA residue”, and a decrease (e.g. about 5.9 fold) for the “SJA residue”; an S2 phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0507] o a decrease (e.g. about 2.6 fold) for the “MAL-II” residue, a decrease (e.g. about 1.4 fold) for the “SNA residue”, a decrease (e.g. about 3.7 fold) for the “GNAresidue”, a decrease (e.g. about 1.6 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 6.8 fold) for the “PNA residue”, and a decrease (e.g. about 13.7 fold) for the “SJA residue”; or

[0508] a G2M S phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:

[0509] o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 3.1 fold) for the “GNA residue”, a decrease (e.g. about 1.6 fold) for the “AAL residue”, a decrease (e.g. about 5.2 fold) for the “STL residue”, a decrease (e.g. about 3.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”.

[0510] Characterising with respect to disease

[0511] A method according to the invention may comprise characterising the particle as being from either a healthy or diseased source.

[0512] Kits of the invention

[0513] In a fifth aspect, the invention provides a kit comprising:

[0514] at least one glycan marker binding partner selected from the group consisting of:

[0515] i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; and iii) AAL or a binding partner for glycan markers having the same specificity as AAL; and at least one binding partner for a second marker selected from the group consisting of:

[0516] • a viability marker;

[0517] • a binding partner for an apoptosis marker.

[0518] A kit according to the fifth aspect of the invention may comprise at least two of the recited glycan binding partners i) to iii), or each of the recited glycan binding partners i) to iii).

[0519] A kit according to any embodiment of the fifth aspect of the invention may comprise each of the recited binding partners for a second marker.A kit according to any embodiment of the fifth aspect of the invention may comprise instructions for its use in a method of the invention for detecting cell death.

[0520] In a sixth aspect, the invention provides a kit comprising:

[0521] at least one glycan marker binding partner selected from the group consisting of:

[0522] i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) NPL or a binding partner for glycan markers having the same specificity as NPL; and iii) MAL II or a binding partner for glycan markers having the same specificity as MAL II; and at least one binding partner for a second marker selected from the group consisting of:

[0523] • a viability marker;

[0524] • a binding partner for an apoptosis marker.

[0525] A kit according to the sixth aspect of the invention may comprise at least two of the recited glycan binding partners i) to iii), or each of the recited glycan binding partners i) to iii).

[0526] A kit according to any embodiment of the sixth aspect of the invention may comprise each of the recited binding partners for a second marker.

[0527] A kit according to any embodiment of the sixth aspect of the invention may comprise instructions for its use in a method of the invention for detecting cell senescence.

[0528] In a seventh aspect the invention provides a kit comprising:

[0529] at least one glycan marker binding partner selected from the group consisting of:

[0530] i) MAL II or a binding partner for glycan markers having the same specificity as MAL II; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; iii) GNA or a binding partner for glycan markers having the same specificity as GNA; iv) AAL or a binding partner for glycan markers having the same specificity as AAL; v) STL or a binding partner for glycan markers having the same specificity as STL; vi) PNA or a binding partner for glycan markers having the same specificity as PNA; and vii) SJA or a binding partner for glycan markers having the same specificity as SJA; and at least one binding partner for a second marker selected from the group consisting of:

[0531] • a viability marker;

[0532] • a binding partner for an apoptosis marker.A kit according the seventh aspect of the invention may comprise at least two of the recited glycan binding partners i) to vii), or at least three of the recited glycan binding partners i) to vii), or at least four of the recited glycan binding partners i) to vii), or at least five of the recited glycan binding partners i) to vii), or at least six of the recited glycan binding partners i) to vii), or each of the recited glycan binding partners i) to vii).

[0533] A kit according to any embodiment of the seventh aspect of the invention may further comprise at least one further reagent selected from the group consisting of: 4',6-diamidino-2-phenylindole (DAPI); a dye for use in mapping cell division number; and an agent for use in analysing cell proliferation. A kit in accordance with this embodiment may comprise at least two of the recited further reagents, or may comprise each of the recited further reagents.

[0534] Suitably in a kit of the seventh aspect of the invention, the dye for use in mapping cell division number may be CellTrace far red (CTFR). Suitably in a kit of the seventh aspect of the invention, the agent for use in analysing cell proliferation is a modified nucleoside (such as 5-ethynyl-2'-deoxyuridine (EDU)).

[0535] Suitably a kit of any aspect seventh aspect of the invention may comprise instructions for its use in a method of the invention for mapping the cell cycle.

[0536] In an eighth aspect, the invention provides a kit comprising:

[0537] at least one glycan marker binding partner selected from the group consisting of:

[0538] i) G.Simp or a binding partner for glycan markers having the same specificity as G.Simp; ii) MAL II or a binding partner for glycan markers having the same specificity as MAL II; iii) SNA or a binding partner for glycan markers having the same specificity as SNA; iv) PNA or a binding partner for glycan markers having the same specificity as PNA; v) SJA or a binding partner for glycan markers having the same specificity as SJA; vi) GNA or a binding partner for glycan markers having the same specificity as GNA; vii) NPL or a binding partner for glycan markers having the same specificity as NPL; viii) Con A or a binding partner for glycan markers having the same specificity as Con A; ix) AAL or a binding partner for glycan markers having the same specificity as AAL; and x) STL or a binding partner for glycan markers having the same specificity as STL; and optionally at least one binding partner for a second marker selected from the group consisting of:

[0539] • a viability marker;

[0540] • a binding partner for an apoptosis marker.Suitably, a kit according to the eighth aspect of the invention may comprise:

[0541] • at least two of the recited glycan binding partners i) to x);

[0542] • at least three of the recited glycan binding partners i) to x);

[0543] • at least four of the recited glycan binding partners i) to x);

[0544] • at least five of the recited glycan binding partners i) to x);

[0545] • at least six of the recited glycan binding partners i) to x);

[0546] • at least seven of the recited glycan binding partners i) to x);

[0547] • at least eight of the recited glycan binding partners i) to x);

[0548] • at least nine of the recited glycan binding partners i) to x); or

[0549] • each of the recited glycan binding partners i) to x).

[0550] A kit according to any embodiment of the eighth aspect of the invention may comprise one of the recited binding partners for a second marker, or may comprise each of the recited binding partners for a second marker.

[0551] Suitably, a kit according to any embodiment of the eighth aspect of the invention comprises instructions for its use in a method of the invention for detecting cell differentiation.

[0552] In a ninth aspect, the invention provides a kit comprising:

[0553] at least one glycan marker binding partner selected from the group consisting of:

[0554] i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) NPL or a binding partner for glycan markers having the same specificity as NPL; iii) Con A or a binding partner for glycan markers having the same specificity as Con A; iv) SNA or a binding partner for glycan markers having the same specificity as SNA; v) MAL II or a binding partner for glycan markers having the same specificity as MAL II; vi) AAL or a binding partner for glycan markers having the same specificity as AAL; vii) UEA-1 or a binding partner for glycan markers having the same specificity as UEA-1 ; viii) LTET or a binding partner for glycan markers having the same specificity as LTET; ix) PNA or a binding partner for glycan markers having the same specificity as PNA; x) SJA or a binding partner for glycan markers having the same specificity as SJA; xi) STL or a binding partner for glycan markers having the same specificity as STL; and xii) G.Simp or a binding partner for glycan markers having the same specificity as G.Simp; and optionally at least one binding partner for a second marker selected from the group consisting of:

[0555] • a viability marker;

[0556] • a binding partner for an apoptosis marker.Suitably, a kit in accordance with the ninth aspect of the invention comprises:

[0557] • at least two of the recited glycan binding partners i) to xii);

[0558] • at least three of the recited glycan binding partners i) to xii);

[0559] • at least four of the recited glycan binding partners i) to xii);

[0560] • at least five of the recited glycan binding partners i) to xii);

[0561] • at least six of the recited glycan binding partners i) to xii);

[0562] • at least seven of the recited glycan binding partners i) to xii);

[0563] • at least eight of the recited glycan binding partners i) to xii);

[0564] • at least nine of the recited glycan binding partners i) to xii);

[0565] • at least ten of the recited glycan binding partners i) to xii);

[0566] • at least eleven of the recited glycan binding partners i) to xii); or

[0567] • each of the recited glycan binding partners i) to xii).

[0568] Suitably, a kit according to any embodiment of the ninth aspect of the invention comprises one of the recited binding partners for a second marker. Suitably, such a kit comprises each of the recited binding partners for a second marker.

[0569] Suitably, a kit according to any embodiment of the ninth aspect of the invention comprises instructions for its use in a method of the invention. Suitably the instructions are for use in a method for detecting cell death (such as detecting apoptosis).

[0570] In a tenth aspect, the invention provides a kit comprising:

[0571] at least one glycan marker binding partner selected from the group consisting of:

[0572] i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; iii) AAL or a binding partner for glycan markers having the same specificity as AAL; iv) PNA or a binding partner for glycan markers having the same specificity as PNA; and v) STL or a binding partner for glycan markers having the same specificity as STL; and optionally at least one binding partner for a second marker selected from the group consisting of:

[0573] • a viability marker;

[0574] • a binding partner for an apoptosis marker.

[0575] In a suitable embodiment, a kit of the tenth aspect of the invention comprises:

[0576] • at least two of the recited glycan binding partners i) to v);at least three of the recited glycan binding partners i) to v);

[0577] at least four of the recited glycan binding partners i) to v); or

[0578] each of the recited glycan binding partners i) to vii).

[0579] Suitably, a kit according to any embodiment of the tenth aspect of the invention comprises one of the recited binding partners for a second marker or comprises each of the recited binding partners for a second marker.

[0580] Suitably, a kit according to any embodiment of the tenth aspect of the invention may comprise instructions for its use in a method of the invention for detecting cell death.

[0581] In a suitable embodiment of a kit of the invention, the viability marker is a binding partner for a nucleic acid. Suitably, such a binding partner for a nucleic acid marker is 7AAD.

[0582] In a suitable embodiment of a kit of the invention, a binding partner for an apoptosis marker is a binding partner for phosphatidylserine. In a suitable example, the binding partner for phosphatidylserine is Annexin V.

[0583] In any suitable kit of the invention, one or more of the binding partners may be labelled with a detection moiety. The detection moiety may be a fluorophore, or a conjugation moiety. Suitably, the conjugation moiety is biotin.

[0584] A kit as herein described may further comprise a detection moiety coupled to a conjugation moiety. Suitably the detection moiety is a fluorophore, such as phycoerythrin. Suitably, the conjugation moiety is streptavidin.

[0585] In an eleventh aspect, the invention provides a kit comprising a binding partner (e.g. lectin) for binding a first glycan biomarker selected from the group consisting of

[0586] i) a terminal sialic acid residue,

[0587] ii) a terminal galactose residue,

[0588] iii) a terminal mannose residue,

[0589] iv) a terminal N-acetylgalactosamine (GalNAc) residue,

[0590] v) a terminal fucose residue,

[0591] vi) a terminal N-acetylglucosamine (GIcNAc) residue, and

[0592] vii) an internal N-acetyllactosamine (LacNAc) residue;and a binding partner for binding a second non-glycan marker, wherein the second marker is selected from the group consisting of: an apoptosis marker; a viability marker; a cell division marker; a cell differentiation marker; a cell cycle marker; a disease marker (whether an acquired disease or an inherited disease); and a cell biology marker.

[0593] In the case of a kit in accordance with this aspect of the invention comprising a lectin as a binding partner for a glycan biomarker, the kit may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more lectin binding partners capable of binding glycan markers. In a suitable embodiment, such a kit of the invention may include twenty or more lectin binding partners capable of binding glycan markers. In a suitable embodiment, such a kit of the invention may include 30 or more, 40 or more, or 50 or more lectin binding partners capable of binding glycan markers. In a suitable embodiment, such a kit of the invention may include 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200 or more lectin binding partners capable of binding glycan markers. By way of example, the lectins may be human lectins. Alternatively, the lectins may be murine lectins.

[0594] In a kit of the invention in accordance with any of the embodiments set out above, at least one of the constituents may be provided in lyophilised form.

[0595] Size Moderation

[0596] Background

[0597] The flow cytometer is an instrument that aligns a cell sample into a single cell stream, to be interrogated by lasers. The laser assesses level of expression of various markers on each cell at a single cell level. In addition, physical qualities of the cell are acquired as special properties of how lasers are interacting with the cell.

[0598] Reference to an “indication” for a marker (whether first, second, third or otherwise) herein preferably refers to a level for the marker (e.g. associated intensity for the marker preferably detected by flow cytometry).

[0599] Forward scatter (FSC) may be captured from a detector that is aligned with a substantially head-on view of the laser beam, while side scatter is captured from a side angle (SSC). These scatters reflect different physical properties of the cell. For example, forward scatter can relateto the cross-sectional area of a cell, while side scatter relates to the complexity or granularity of a cell. The detection of the laser interaction with cells is recorded by the cytometer.

[0600] In an ideal world, fluorescence have narrow emission ranges such that they do not complicate other channels. However, in reality, there is significant “spillage” from one channel to the next, especially if the emission wavelength are close together. Since the dawn of the flow cytometer’s invention in 1970s, hardware and software has been orientated to solve a compensation issue due to adjacent channel’s signals spilling into each other. A mathematical fix was provided to compensate for this spillage.

[0601] In particular, the contribution from outside channels is removed. To work out how much to remove, compensation controls are applied by first removing the signal in question (red for example), and asking how much another signal (such as green) is contributing. This results in a compensation percentage to permit calculation of compensated fluorescence.

[0602] The flow cytometer data also typically undergoes gating. Many gates, which are methods of ring fencing a subset of data points from a mother population to produce a subpopulation, are applied to flow data to end up with a desired subset for final analysis and presentation. The first three gates applied often comprise: a) cell gate, b) single cell gate, and c) live cell gate. Gating essentially provides polygons, rectangle, and circle drawing tools to allow users to craft the gates for selection of the subpopulations from the mother populations.

[0603] The compensated data, in a gated subpopulation, may then be used for analysis, typically presented in either 1D (histograms) or 2D plots.

[0604] Certain embodiments involve determining the “density” of the glycan first marker on the external surface of the particle (e.g. based on the surface area of the external surface). Similarly, certain embodiments involve determining the “density” of the second marker on the external surface of the particle. Such embodiments are particularly relevant when the markers are determined using flow cytometry.

[0605] “Density” of a marker as claimed may be determined as follows via “moderation” steps. Thus, as a synonymous term to “density”, it may be said that methods of the invention make use of a “moderated level” for the glycan marker and / or a “moderated” level for the second marker. The moderation steps may be conducted as steps of the claimed methods. That being said, it should be appreciated that methods of the invention can make use of ‘already moderated’ levels, in which this moderation has been conducted previously and the moderated levels then imported to the claims. As such that, these following moderation steps discussed below can be used to define the term “density” in the context of the present disclosure, with reference to“how” density is calculatable (e.g. without ‘active’ moderation steps being limiting on the claims).

[0606] Moderating the indication (aka level) of the marker present for one or more particles within the sample based on an indication of size may be particularly advantageous to experimental scenarios where particle sizes are changing. These scenarios include common cell biology events such as death, aging, cycling, development, and division, where cells may change by factors of approximately 2-3. Even more extreme scenarios might be production of extracellular microvesicles or nanovesicles where particles with more than 10-fold size reduction are produced, and in large quantities. The inventors have identified this problem that has, until now, been overlooked since the inception of flow cytometry. Upon reflection, it is in fact critical to moderate flow cytometry data based on size, and its lack of implementation has already likely caused errors in 50 years of flow cytometry history.

[0607] By using the forward scatter, a mathematical fix can be applied. Forward scatter (FSC) may be taken to approximate cross-sectional area, which is proportional to the cell surface area. As such, dividing by FSC provides a good approximation of the surface density of a marker. Similarly, for an internal marker, division by FSC3 / 2, provides a good approximation of volumetric density of a marker.

[0608] As such, moderating the indication of the marker present for one or more particles (such as, but not limited to one or more cells, vesicles, and / or specific cell bodies) within the sample using the forward scatter signal may comprise dividing the indication of the marker present based on the forward scatter signal to determine an indication of density of the marker for the one or more particles, for example, by (i) dividing the indication of the marker present by the forward scatter signal to determine an indication of surface density of the marker for the one or more particles (for example wherein the indication of the marker present comprises an indication of a surface-binding marker), and / or (ii) dividing the indication of the marker present by FSC3 / 2to determine an indication of volumetric density of the markerwithin the one or more particles (for example wherein the indication of the marker present comprises an indication of an intracellular marker).

[0609] The method may comprise moderating the flow cytometry data for one particle by dividing the indication of the marker present in said one particle based on the forward scatter signal for said one particle. This may be advantageous to moderate the flow cytometry data for individual cells, or biological matter (such as vesicles and / or specific cell bodies etc), and can assist visualisation in 1 dimensional or multi-dimensional representations. Said moderation can also be applied to high dimensional analysis, such as principal component analysis (PCA) and t-stochastic neighbour embedding (tSNE).Alternatively, the method may comprise moderating the flow cytometry data for a plurality of particles by determining an average indication of the marker present for said plurality of particles; determining an average forward scatter signal for said plurality of particles; and dividing the average indication of the marker present for said plurality of particles based on the average forward scatter signal for said plurality of particles. The moderation for size could be applied to a population or subpopulation, for example focusing on the average fluorescence of a group of particles, such as cells / vesicles etc. The method may also comprise gating the flow cytometer data to identify the population of particles (e.g. cells, vesicles, and / or specific cell bodies etc) within the sample.

[0610] The indication of the marker present for one or more cells or particles (such as, but not limited to vesicles, and / or specific cell bodies) within the sample may comprise an indication of fluorescence of the one or more particles within the sample, wherein said marker is configured to fluoresce.

[0611] The indication of the marker present may comprise an indication of a molecule present in the one of more particles, such as but not limited to a sugar, glycan, lipid, DNA, RNA, or protein. The marker may be a biomarker or a reporter, for example. Applying the method using markers configured to provide an indication of a ubiquitous molecule, such as sugars (e.g. a glycan described herein), may provide some advantages over selecting a specific marker, such as a specific protein for example. A ubiquitous molecule may be a molecule that is present on substantially all cell particles, preferably including vesicles. For example, specific markers, such as specific protein markers, may only be used for cells that have said specific marker on the surface and therefore cannot be used to normalise between cells in a sample which do not have the specific protein. By contrast, markers for ubiquitous molecules, such as sugars (including, but not limited to, terminal sugar or sub terminal sugar residues, cell-surface or intracellular glycoconjugates, and optionally glycans), may be detected across substantially all cell types and states. In other words, moderating for ubiquitous molecules, for example using sugar-recognising markers, may enable differentiation between any cells or particles, or any ‘population’ thereof, in a generalised or ubiquitous way. This is not possible when using markers configured for specific molecules, such as specific protein recognition on the cell surface.

[0612] The method may also comprise obtaining the flow cytometer data for a sample, wherein the flow cytometer data comprises indications of two or more markers present for one or more particles within the sample, and a FSC for each of the particles. The two or more markers may be configured to provide an indication of the presence of different molecules. The method may then comprise moderating the indication of each marker present for the particles within thesample based on the indication of size for said particles using the forward scatter signal. Moderating indications from two or more molecules on the same particle may be advantageous for confirming the same particle from two or more signals for internal consistency and signal confirmation. Preferably, at least one of the markers is configured to provide an indication of a ubiquitous molecule, such as a sugar molecule. In some examples, a plurality of markers may be configured to provide an indication of a ubiquitous molecule, such as a plurality of different sugars, for example. This may be advantageous in comparison to methods utilising single markers, not least because the signals can be compared against particles which do not necessarily present said specific single marker.

[0613] As noted above, in some examples, the method may be applied for sugar signal moderation, including for example moderation of terminal sugar signals or sub terminal sugar residues, for the purpose of, for example, cell-state determination.

[0614] Sugar signal moderation may provide a ubiquitous method of differentiating between any and / or all cells in a population. The method may also be suitable for use with live cells. Lectin-based markers may be advantageous for providing an indication of a sugar (aka glycan herein) molecule present, including for example terminal sugar molecules. However, the skilled person will understand that other known sugar-recognising marker systems may be used, including for example but not limited to other carbohydrate-binding proteins, antibodies, and other binding partners described herein.

[0615] For example, a selection of non-limiting examples have been included below.

[0616] (i) the indication of the marker may comprise an indication of Annexin V, wherein moderating the flow cytometry data comprises dividing the indication of Annexin present by the forward scatter signal to determine an indication of surface density of Annexin for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc). This may be particularly advantageous for apoptosis analysis.

[0617] (ii) the indication of the marker may comprise an indication of 7-AAD, wherein moderating the flow cytometry data comprises dividing the indication of 7-AAD present by FSC3 / 2to determine an indication of volumetric density of 7-AAD for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc). This may be particularly advantageous for necrosis analysis.

[0618] (iii) the indication of the marker may comprise an indication of lectin, wherein moderating the flow cytometry data comprises dividing the indication of lectin present by the forward scatter signal to determine an indication of surface densityof lectin for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc).

[0619] The indication of the marker present for one or more particles within the sample may comprise an indication of a number of the marker present for the one or more particles within the sample. In some examples, the method may further comprise obtaining flow cytometer data for a control sample, the flow cytometer data comprising a forward scatter signal, FSC, for each one or more particles within the control sample, wherein the one or more particles in the control sample are of known size, for example wherein the one or more particles may comprise sizing beads. The method may then determine size calibration data which associates FSC signals with a particle size, based on the obtained flow cytometer data for the control sample in view of the known size and obtained FSC signal. In such examples, moderating the indication of the marker present for one or more particles within the sample based on an indication of size for said one or more particles may use the forward scatter signal and the size calibration data. This may be advantageous to calibrate the analysis for a certain flow cytometer and its operating parameters in order to establish the relationship between actual size of a particle and FSC signal, rather than merely relative or proportional size of a particle.

[0620] For example, moderating the indication of the marker present for one or more particles within the sample may comprise determining an indication of size for said one or more particles using the forward scatter signal for said one or more particles and the size calibration data; and dividing the indication of the marker present based on the indication of size to determine an indication of density of the marker for the one or more particles of the sample. As above, dividing the indication of the marker present based on the indication of size may comprise: (i) dividing the indication of the marker present by the indication of size to determine an indication of surface density (sDEN) of the marker for the one or more particles; and / or (ii) dividing the indication of the marker present by the indication of size to the power of 3 / 2, (the indication of size)372, to determine an indication of volumetric density (vDEN) of the marker within the one or more particles.

[0621] The skilled person will understand that references to particles, cells, vesicles, and / or specific cell bodies etc within the sample are not intended to be limiting and that the method of the present invention may be applied to any biological particles, bodies, or otherwise biological matter, within the biological sample for which flow cytometry can be performed. In addition, the skilled person will also understand that the present method may also be applied to non-biological particles and entities, such as but not limited to beads (for example such as beads bound or dosed with a marker configured to be detected by a flow cytometer).Another aspect of the invention provides for a computer-implemented method for graphical analysis of flow cytometry data analysis. The method comprises performing the method for moderating flow cytometry data of the preceding aspect, and graphically plotting the moderated indication of the marker present for one or more particles within the sample. This may be particularly advantageous for improving identification of populations and / or subpopulations within the sample.

[0622] Graphically plotting the moderated indication of the marker present may comprise plotting the moderated indication of the marker present for the one or more particles within the sample on a first axis, and plotting a moderated indication of a second marker present for the same one or more particles within the sample on a second axis. For example, wherein the moderated indication of the second marker is obtained in the same way as described above.

[0623] In some examples, graphically plotting the moderated indication of the marker present may comprise plotting lectin density data for the one or more particles within the sample on a first axis, and plotting the moderated indication of the marker present for the same one or more particles within the sample on a second axis. This may be particularly advantageous because the combination of lectin data, in combination with other markers, enables improved analysis as the lectin data provides extra dimensions of information beyond conventional markers alone.

[0624] For example, the indication of the marker present may comprise an indication of lipids present. Graphically plotting the moderated indication of the marker present may then comprise plotting lectin density data for the one or more particles within the sample on the first axis, and plotting the moderated lipid density data for the same one or more particles within the sample on the second axis.

[0625] Whilst the above disclosure discusses using the forward scatter signal to moderate the indication of the marker present based on an indication of size for said one or more particles, the skilled person will understand that this is not limiting, and that indication of size may be determined in other ways. As such, another aspect of the invention provides for a computer-implemented method for moderating flow cytometry data for a sample, the method comprising:

[0626] obtaining flow cytometer data for a sample, the flow cytometer data comprising an indication of a marker present for one or more particles within the sample;

[0627] obtaining an indication of size for the one or more particles; and

[0628] moderating the indication of the marker present for one or more particles within the sample by the indication of size.In some examples, the indication of size may be obtained using image analysis of at least one image of the one or more particles within the sample. For example, the flow cytometer data for the sample may be imaging flow cytometer data comprising at least one image of the one or more particles within the sample. Obtaining the indication of size for the one or more particles within the sample may then comprise determining cell size based on image analysis of the at least one image. By way of non-limiting example, image analysis may comprise counting image pixels or any other suitable way of determining size based on image analysis, for example including Al-based techniques.

[0629] In some examples, the indication of size may be obtained based on a combination of image analysis and the FSC analysis discussed above. This may improve accuracy and reliability of the indication of size.

[0630] Alternatively, or in addition, the flow cytometer data may further comprise electrical impedance data for the one or more particles within the sample, wherein obtaining the indication of size for the one or more particles is based on the electrical impedance data.

[0631] In some examples, the indication of size may be obtained based on a combination of electrical impedance analysis and the FSC analysis discussed above. This may improve accuracy and reliability of the indication of size.

[0632] Another aspect of the invention provides for a computer-implemented method of identifying cell subpopulations within a biological sample. The method comprises obtaining flow cytometer data for a population of particles (such as, but not limited to cells, vesicles, and / or specific cell bodies) within a biological sample. The flow cytometer data comprises a first indication of a first marker present for each particle within the population of particles, and a second indication of a second marker present for each particle within the population of particles. Optionally the second indication is obtained based on at least one lectin marker. The method then comprises obtaining an indication of size for each of the particles within the population, and moderating (i) the first indication of the first marker present for each particle, and (ii) the second indication of the second marker present for each particle, using indication of size for said particle.

[0633] The present method may be advantageous in order to scientifically quantify changes in surface expression of glycan classes, and / or other molecules, by normalising against size. For example, cell size changes greatly during the birth of apoptotic bodies. This method may therefore facilitate quantification of changes in surface expression of molecules, such as glycans, and enable comparison between cells and apoptotic bodies, by normalising againstsurface area. This method may , in some examples, unify analysis of Lectome density across all cells and vesicles with varying cell size.

[0634] The method may then further comprise graphically plotting the moderated indication of the first marker on a first axis, and the moderated indication of the second marker on a second axis. A first subpopulation of particles (such as cells, vesicles, and / or specific cell bodies) within the population may then be identified based on the graphical plot.

[0635] In some examples, the first indication of the first marker may comprise an indication of lipids present for each cell within the population of particles, for example but not limited to an indication of phosphatidylserine present for each particle within the population. For example, an indication phosphatidylserine may be obtained based on an Annexin marker.

[0636] The second indication of the second marker may comprise an indication of glycans present for each cell within the population of cells, vesicles, and / or specific cell bodies based on at least one lectin marker. Alternatively or in addition, other terminal sugar recognising molecule I markers may be used.

[0637] Aspects of the present invention therefore aim to provide a novel approach to be able to compare distinct cellular populations, for example based on analysis of DNA content, surface lipid and glycans. However, the skilled person will understand the same methods easily extend to other analyses, such as but not limited to protein content and surface expression normalisation, indeed it may be applied for any marker that can be detected by a flow cytometer. The skilled person will also understand that the same methods may also extend to comparison between cells / particles, for example for population to population and / or particle to particle comparisons.

[0638] In some examples, the flow cytometer data further comprises a forward scatter signal, FSC, for each particle, wherein the indication of size is obtained based on the forward scatter signal for each particle, for example as was described in the first aspect of the invention.

[0639] Alternatively, or in addition, the indication of size may be obtained using image analysis of at least one image of the one or more particles within the sample. For example, the flow cytometer data for the sample may be imaging flow cytometer data comprising at least one image of the one or more particles within the sample. Obtaining the indication of size for the one or more particles within the sample may then comprise determining particle size based on image analysis of the at least one image. Byway of non-limiting example, image analysis may comprise counting image pixels or any other suitable way of determining size based on image analysis, for example including Al-based techniques.Alternatively, or in addition, in some examples, the flow cytometer data may further comprise electrical impedance data for the one or more particles within the sample, wherein obtaining the indication of size for the one or more particles is based on the electrical impedance data. Incorporation of size moderation, in particular based on surface area and / or volume normalisation, to basic flow cytometer output may be advantageous universally across many different areas of cell biology, cell cycle, stem cell differentiation, and development, etc. As one example, the method may be advantageous for analysing the cell death programme, for example analysing exit of apoptosis into necrosis. Size moderation as disclosed herein may be particularly effective for cell death analysis because the cell death process contains substantial cell changes, such as when cells reduce in size as apoptotic bodies are formed, and further reduction in size when microvesicles are formed.

[0640] However, the skilled person will understand that substantial size changes are not unique to the cell death programme, and as such the method may also have advantageous applications for other biological analyses.

[0641] The viability marker may comprise, but is not limited to, at least one of:

[0642] (i) Annexin V, and wherein identifying the first subpopulation of particles based on the corrected indication of the Annexin marker comprises identifying the first subpopulation of particles to be dead by apoptosis; or

[0643] (ii) 7-AAD, and wherein identifying the first subpopulation of particles based on the corrected indication of the 7-AAD marker comprises identifying the first subpopulation of particles to be dead by necrosis.

[0644] In some examples, the flow cytometer data further comprises a forward scatter signal, FSC, for each particle, wherein the indication of size is obtained based on the forward scatter signal for each particle, for example as was described in the first aspect of the invention.

[0645] Alternatively, or in addition, the indication of size may be obtained using image analysis of at least one image of the one or more particles within the sample. For example, the flow cytometer data for the sample may be imaging flow cytometer data comprising at least one image of the one or more particles within the sample. Obtaining the indication of size for the one or more particles within the sample may then comprise determining cell size based on image analysis of the at least one image.Alternatively, or in addition, in some examples, the flow cytometer data may further comprise electrical impedance data for the one or more particles within the sample, wherein obtaining the indication of size for the one or more particles is based on the electrical impedance data. Another embodiment of the invention provides for a graphical representation of a plurality of marker for a cell or vesicle comprising:

[0646] a plurality of circular sectors arranged around a central point, wherein each sector represents one marker such that the plurality of sector collectively represent a plurality of markers; and

[0647] wherein the radius of each circular sector from the central point is proportional to a density of the respective marker present for the cell or vesicle.

[0648] For example, the plurality of markers may comprise one or more surface lectins or other terminal sugar recognising molecule I marker.

[0649] Each circular sector may be assigned a colour, wherein the colour represents a type of marker or lectin.

[0650] Each circular sector may further be assigned a shade of a colour, wherein each shade represents a sub-type of marker or lectin (e.g. a letter in the Lectomic code, for example sialic acids, either alpha 2,6 linked or alpha 2,3 linked).

[0651] More specific description of moderation

[0652] While fluorescence compensation is a major part of data analysis in flow cytometry, compensation, moderation, or normalisation of flow cytometry data based on cell or particle size has been absent historically since the advent of flow cytometry analysis.

[0653] The general principle is that since cell size changes during normal cell biological processes -such as apoptosis, senescence, and cell cycling, as well as specialised situations such as T cell activation - flow cytometric analysis has forgotten about an important normalisation of cell surface markers and intracellular markers. The omission of accounting for dynamic size changes is also particularly important for the formation of extracellular vesicles which can be up to ten times smaller than cells.

[0654] The problem arising from existing flow cytometry techniques which do not moderate based on size is illustrated in a schematic Fig. 28.

[0655] Purely for illustration, (A) depicts a cell 100 comprising eight surface markers 102. Each marker is marked with a marker (for example such as a fluorescent marker) such that it gives rise to a signal during flow cytometry.As a result of some biological process, cell 100 reduces in size. As shown in (B), whilst the cell has reduced in size, it maintains its eight surface markers 102. As such, in traditional flow cytometry analysis, the signal quantity would be the same as for the larger cell 100 shown in (A). This leads to the analysis conclusion, based on raw flow cytometer data, that nothing has changed, regardless of the significant size reduction and increase in surface density which is not reflected within the “raw” flow cytometry data. Clearly, the cells shown in (A) and (B) cannot meaningfully be compared on the basis of the raw flow cytometry data.

[0656] By contrast, (C) illustrates a schematic where the flow cytometer signal has been moderated proportionally according to cell size. In such an example, a proportional signal reduction is observed. Moderation according to size therefore assists in identification of changes in cell populations.

[0657] FIG. 29 shows a box diagram of an example system 200 of the invention. The system 200 comprises a flow cytometer 202, a processor 204, and optionally a display 206.

[0658] The flow cytometer 202 is configured to generate flow cytometry data based on a sample. The flow cytometer data comprises an indication of a marker present within the sample, and a forward scatter signal (FSC) for particles, such as cells, vesicles, specific cell bodies, or other biological particles, within the sample.

[0659] The processor 204 is configured to receive the flow cytometry data collected by the flow cytometer 202, and then to moderate the flow cytometer data based on an indication of size using the forward scatter signal.

[0660] In this example, two primary types of moderation are provided: (i) surface density normalisation (sDEN), and (ii) volumetric density normalisation (vDEN). These are discussed in more detail below.

[0661] Fig. 30 shows a flow diagram of an example method of the invention, for example for use with the system of Fig. 29.

[0662] Firstly, the method comprises obtaining flow cytometer data for a biological sample (310). The flow cytometer data comprises an indication of the presence of a marker within the sample, and a forward scatter signal, FSC. The flow cytometer data may be obtained at a processor 204 from a flow cytometer 202. The indication of the presence of a marker within the sample may comprise a signal indicative of fluorescence for a plurality of cells or biological bodies (such as vesicles, cell bodies, or other particles) within the sample, wherein the sample has been marked with a fluorescent stain configured to bind to a biological molecule of the cell or particle.Secondly, the method comprises moderating the indication of the marker present within the sample based on an indication of size of the cell or particle using the forward scatter signal. This step may be performed by processor 204. The term “moderation” is used to refer to a normalisation step which intends to correct the impact of size on the flow cytometer signal relating to the expression of markers.

[0663] Two primary types of moderation are provided: (i) surface density normalisation (sDEN), and (ii) volumetric density normalisation (vDEN). These are discussed in more detail below. Moderation may be applied on an individual cell I particle basis, or on a population basis. This is also discussed in more detail below.

[0664] The processor 204 may then use the moderated flow cytometer data for the purpose of sample gating, for example wherein the step of moderation 320 is performed prior to gating of the sample.

[0665] Optionally, the processor 204 may graphically plot the moderated indication of the marker present for one or more cells and / or particles within the sample.

[0666] The processor 204 may then send the moderated flow cytometer data to a display device 206, wherein the display device is configured to display it to a user.

[0667] Furthermore, the processor 204 is preferably configured to identifying at least one population or subpopulation of cells or other biological bodies I particles within the sample based on the moderated indication of the markers present.

[0668] Surface density normalisation, sDEN

[0669] This moderation equation addresses the surface marker’s relationship to the surface area of a cell.

[0670] The density of the marker e.g. glycan marker (or moderated level for the marker e.g. glycan marker) may mean surface density.

[0671] Any reference to a level of a marker described herein may mean a density (e.g. surface or volumetric density) obtainable by a moderation step described herein.

[0672] As an approximation, the forward scatter (FSC) from the flow cytometer data is taken to be proportional to the cross-sectional area of a passing cell (see formula i below).

[0673] FSC oc cross sectional area = nr2The particles (e.g. cells and / or vesicles) are approximated to have a surface area of an assumed sphere (A= 4nr2, meaning surface area is directly proportional to cross-sectional area. It therefore follows that FSC is directly proportional to surface area.

[0674] This provides formula ii:

[0675] Signal

[0676] sDEN surface density) = ks

[0677] FSC

[0678] wherein ksis a constant and can be ignored when comparing between cell or other particles. Thus, surface density for a marker described herein (e.g. of a glycan first marker and / or second marker described herein) may be a level for said marker that has been moderated by formula ii.

[0679] For a population or subpopulation (formula iii):

[0680] population average (e. g. fluorescence) signal

[0681] sDEN

[0682] population average surface area

[0683] For example (formula iv):

[0684] population average signal (e. g. fluorescence)

[0685] sDEN = - - - — — - - - population average FSC — A

[0686] where FSC-A is the area of the forward scatter signal. A similar approximation may be derived from FSC-H, the height of the forward scatter signal. It is preferred to use FSC-A as more information about the forward scatter is captured.

[0687] Thus, surface density for a marker described herein (e.g. of a glycan first marker and / or second marker described herein) may be a level for said marker that has been moderated by formula iii. Additionally or alternatively, surface density for a marker described herein (e.g. of a glycan first marker and / or second marker described herein) may be a level for said marker that has been moderated by formula iv.

[0688] For example, if marker A is measured with the fluorochrome, FITC, population average FITC — A

[0689] sDEN = - - - — — - - population average FSC — A

[0690] where “average” could be mean fluorescence intensity or geometric fluorescence intensity. Any other employed detection moiety (e.g. fluorochrome other than FITC) may substitute for FITC in the formula above.At a single cell level,

[0691] single cell fluorescence

[0692] sDEN =

[0693] single cell surface area

[0694] As such, if marker A is measured with the fluorochrome, FITC,

[0695] single cell FITC — A

[0696] sDEN =

[0697] single cell FSC — A

[0698] One dimensional histograms, 2D and higher dimensional representations could represent these new surface area compensated data points. These single cell or population averaged surface densities could be applied to hyper dimensional reduction calculations, such as tSNE or PCA.

[0699] Volumetric density normalisation, vDEN

[0700] The density of a marker (or moderated level for the marker), such as the second marker, may mean volumetric density.

[0701] When considering intracellular markers (e.g. as the second marker), the calculation of density may need to be adjusted to account for cellular volume rather than cross-sectional or surface area. Since,

[0702] 4

[0703] Volume = — nr

[0704] 3

[0705]

[0706] This provides (formula v):

[0707] Signal

[0708] vDEN (volumetric density) = kv- j

[0709] (FSC)2

[0710] wherein kvis a constant and can be ignored when comparing between cell or other particles. Thus, volumetric density for a marker described herein (e.g. second marker described herein) may be a level for said marker that has been moderated by formula v.

[0711] For the population (formula vi),

[0712] population average of signal (e. g. fluorescence)

[0713] vDEN = - 3 - (population average cross sectional ared)2population average of signal (e. g. fluorescence')=3

[0714] (population average FSC)2

[0715] Thus, volumetric density for a marker described herein (e.g. second marker described herein) may be a level for said marker that has been moderated by formula vi.

[0716] For the single cell (formula vii),

[0717] sinqle cell fluorescence

[0718] vDEN = - - - - - 3

[0719] (single cell surface area)2

[0720] single cell fluorescence

[0721] =3

[0722] (single cell FSC)2

[0723] Thus, volumetric density for a marker described herein (e.g. second marker described herein) may be a level for said marker that has been moderated by formula vii.

[0724] The size normalisation provides an advantageous type of fundamental normalisation I moderation of flow cytometry analysis, that has been omitted since the invention of flow cytometry.

[0725] Whether moderation is performed according to surface density normalisation or volumetric density moderation ultimately depends on the marker being investigated and its cellular expression, for example if the marker being investigated demonstrates a surface expression, surface density normalisation is applied; whereas if the marker being investigated demonstrated intracellular expression, volumetric density normalisation is applied.

[0726] Merely by way of example:

[0727] • DNA’. If DNA is being investigated, for example using an intracellular marker such as 7AAD, moderating the indication of 7AAD I DNA provided by the flow cytometer may be defined by:

[0728] Raw 7AAD

[0729] DNA content Density [7AAD] = - 3

[0730] (FSC - A 2

[0731] Glycan (e.g. Lectin or other suitable binding partner for a glycan) markers’. If cellsurface glycan (e.g. lectin) markers are used, moderating such may comprise:Raw glycan (e. g. lectin) signal

[0732] Glycan (e. g. Lectomic) Density = - — — - - -

[0733] Alternatively, the skilled person will understand that if intracellular markers (e.g. second markers) are used, moderating such may comprise:

[0734] Raw intracellular marker signal intracellular marker Density = - - (FSC - A 2

[0735] For example, the skilled person will understand that if intracellular glycan (e.g. lectin) markers are used, moderating such may comprise:

[0736] Raw glycan (e. g. Lectomic) signal Glycan (e. g. Lectomic) Density = - - (FSC - A 2

[0737] • Phosphatidylserine, PS (e.g. Annexin Vf. If phosphatidylserine (e.g. Annexin V) markers are used, moderating such may comprise:

[0738] Raw PS e. g. AnnexinV

[0739] PS (e. g. AnnexinV) Density = - ~FSC — A -

[0740] In addition to the above, the skilled person will also understand that specific marker density may be determined based on the change in the indication of a marker present from a sample, relative to a control sample.

[0741] For example,

[0742] A glycan binding partner (e. g. lectin) signal Specific glycan (e. g. lectin) Density = - — — - - - FSC — A

[0743] and / or

[0744] A PS (e. g. AnnexinV) binding Specific PS (e. g. AnnexinV) Density = - — - - - r /

[0745] where A glycan (e.g. Lectin) or A PS (e.g. Annexin-V) binding are calculated by subtracting glycan (e.g. Lectin) or PS (e.g. Annexin-V) control binding from respective raw binding signals. However, the skilled person will understand that this is not limited to only glycan (e.g. lectin) or PS (e.g. Annexin-V) markers, and that the same may be calculated for other markers.The flow cytometer is an instrument that aligns a cell sample into a single cell stream, to be interrogated by lasers. The laser assesses level of expression of various markers on each cell at a single cell level. In addition, physical qualities of the cell are acquired as special properties of how lasers are interacting with the cell. Forward scatter (FSC) may be captured from a detectorthat is aligned with a substantially head-on view of the laser beam, while side scatter is captured from a side angle (SSC). These scatters reflect different physical properties of the cell. For example, forward scatter can relate to the cross-sectional area of a cell, while side scatter relates to the complexity or granularity of a cell. The detection of the laser interaction with cells is recorded by the cytometer.

[0746] In an ideal world, fluorescence have narrow emission ranges such that they do not complicate other channels. However, in reality, there is significant “spillage” from one channel to the next, especially if the emission wavelength are close together. Since the dawn of the flow cytometer’s invention in 1970s, hardware and software has been orientated to solve a compensation issue due to adjacent channel’s signals spilling into each other. A mathematical fix was provided to compensate for this spillage. In particular, the contribution from outside channels is removed. To work out how much to remove, compensation controls are applied by first removing the signal in question (red for example), and asking how much another signal (such as green) is contributing. This results in a compensation percentage to permit calculation of compensated fluorescence. The flow cytometer data also typically undergoes gating. Many gates, which are methods of ring fencing a subset of data points from a mother population to produce a subpopulation, are applied to flow data to end up with a desired subset for final analysis and presentation. The first three gates applied often comprise: a) cell gate, b) single cell gate, and c) live cell gate. Gating essentially provides polygons, rectangle, and circle drawing tools to allow users to craft the gates for selection of the subpopulations from the mother populations.

[0747] The compensated data, in a gated subpopulation, may then be used for analysis, typically presented in either 1D (histograms) or 2D plots.

[0748] Determining density via a moderation step may involve flow cytometer data for a sample, the flow cytometer data comprising an indication of a marker present for one or more particles within the sample, and a forward scatter signal, FSC, for each of the one or more particles. The step of “determining the presence of a glycan first marker” and / or the step of “determining the presence of a second marker” provides said indication of a marker present for one or more particles.The indication of the marker present for one or more particles (e.g. within the sample) may then be moderated based on an indication of size for said one or more particles using the forward scatter signal.

[0749] Moderating the indication of the marker present for one or more particles within the sample based on an indication of size may be particularly advantageous to experimental scenarios where particle sizes are changing. These scenarios include common cell biology events such as death, aging, cycling, development, and division, where cells may change by factors of approximately 2-3. Even more extreme scenarios might be production of extracellular microvesicles or nanovesicles where particles with more than 10-fold size reduction are produced, and in large quantities. The inventors have identified this problem that has, until now, been overlooked since the inception of flow cytometry. Upon reflection, it is in fact notably advantageous to moderate flow cytometry data based on size.

[0750] By using the forward scatter, a mathematical fix can be applied. Forward scatter (FSC) may be taken to approximate cross-sectional area, which is proportional to the cell surface area. As such, dividing by FSC provides a good approximation of the surface density of a marker. Similarly, for an internal marker, division by FSC3 / 2, provides a good approximation of volumetric density of a marker.

[0751] As such, moderating the indication of the marker present for one or more particles (as described herein) within the sample using the forward scatter signal may comprise dividing the indication of the marker present based on the forward scatter signal to determine an indication of density of the marker for the one or more particles, for example, by (i) dividing the indication of the marker present by the forward scatter signal to determine an indication of surface density of the marker for the one or more particles (for example wherein the indication of the marker present comprises an indication of a surface-binding marker), and / or (ii) dividing the indication of the marker present by FSC3 / 2to determine an indication of volumetric density of the marker within the one or more particles (for example wherein the indication of the marker present comprises an indication of an intracellular marker).

[0752] Flow cytometry data for one particle may be moderated by dividing the indication of the marker present in said one particle based on the forward scatter signal for said one particle. This may be advantageous to moderate the flow cytometry data for individual particles (e.g. individual cells, or vesicles and / or specific cell bodies etc), and can assist visualisation in 1 dimensional or multi-dimensional representations. Said moderation can also be applied to high dimensional analysis, such as principal component analysis (PCA) and t-stochastic neighbour embedding (tSNE).That being said, the flow cytometry data for a plurality of particles may be moderated by determining an average indication of the marker present for said plurality of particles; determining an average forward scatter signal for said plurality of particles; and dividing the average indication of the marker present for said plurality of particles based on the average forward scatter signal for said plurality of particles. The moderation for size could be applied to a population or subpopulation, for example focusing on the average fluorescence of a group of particles, such as cells / vesicles etc. The method may also comprise gating the flow cytometer data to identify the population of particles (e.g. cells, vesicles, and / or specific cell bodies etc) within the sample.

[0753] The indication of the marker present for one or more cells or particles (such as, but not limited to vesicles, and / or specific cell bodies) within the sample may comprise an indication of fluorescence of the one or more particles within the sample, wherein said marker is configured to fluoresce.

[0754] The indication of the marker present may comprise an indication of a molecule present in the one of more particles, such as but not limited to a sugar, glycan, lipid, DNA, RNA, or protein. The marker may be a biomarker or a reporter, for example.

[0755] For example, a selection of non-limiting examples have been included below.

[0756] (i) the indication of the marker may comprise an indication of phosphatidylserine (e.g.

[0757] via an Annexin binding / detection partner), wherein moderating the flow cytometry data comprises dividing the indication of phosphatidylserine present by the forward scatter signal to determine an indication of surface density of phosphatidylserine (e.g. Annexin V) for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc). This may be particularly advantageous for apoptosis analysis.

[0758] (ii) the indication of the marker may comprise an indication of a nucleic acid stain for example as the second marker (e.g. via a 7-AAD binding / detection partner), wherein moderating the flow cytometry data comprises dividing the indication of the nucleic acid stain (e.g. 7-AAD) present by FSC3 / 2to determine an indication of volumetric density of the nucleic acid stain (e.g. 7-AAD) for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc). This may be particularly advantageous for necrosis analysis.

[0759] (iii) the indication of the marker may comprise an indication of glycan binding partner (e.g. a lectin), wherein moderating the flow cytometry data comprises dividing the indication of the glycan binding partner (e.g. lectin) present by the forward scattersignal to determine an indication of surface density of the glycan binding partner (e.g. lectin) for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc).

[0760] The indication of the marker present for one or more particles within the sample may comprise an indication of a number of the marker present for the one or more particles within the sample. In some examples, the method may further comprise obtaining flow cytometer data for a control sample, the flow cytometer data comprising a forward scatter signal, FSC, for each one or more particles within the control sample, wherein the one or more particles in the control sample are of known size, for example wherein the one or more particles may comprise sizing beads. The method may then determine size calibration data which associates FSC signals with a particle size, based on the obtained flow cytometer data for the control sample in view of the known size and obtained FSC signal. In such examples, moderating the indication of the marker present for one or more particles within the sample based on an indication of size for said one or more particles may use the forward scatter signal and the size calibration data. This may be advantageous to calibrate the analysis for a certain flow cytometer and its operating parameters in order to establish the relationship between actual size of a particle and FSC signal, rather than merely relative or proportional size of a particle.

[0761] For example, moderating the indication of the marker present for one or more particles within the sample may comprise determining an indication of size for said one or more particles using the forward scatter signal for said one or more particles and the size calibration data; and dividing the indication of the marker present based on the indication of size to determine an indication of density of the marker for the one or more particles of the sample. As above, dividing the indication of the marker present based on the indication of size may comprise: (i) dividing the indication of the marker present by the indication of size to determine an indication of surface density (sDEN) of the marker for the one or more particles; and / or (ii) dividing the indication of the marker present by the indication of size to the power of 3 / 2, (the indication of size)372, to determine an indication of volumetric density (vDEN) of the marker within the one or more particles.

[0762] Another aspect of the invention provides for a computer-implemented method for graphical analysis of flow cytometry data analysis. The method comprises performing the method for moderating flow cytometry data of the preceding aspect, and graphically plotting the moderated indication of the marker present for one or more particles within the sample. This may be particularly advantageous for improving identification of populations and / or subpopulations within the sample.Graphically plotting the moderated indication of the marker present may comprise plotting the moderated indication of the marker present for the one or more particles within the sample on a first axis, and plotting a moderated indication of a second marker present for the same one or more particles within the sample on a second axis. For example, wherein the moderated indication of the second marker is obtained in the same way as described above.

[0763] In some examples, graphically plotting the moderated indication of the marker present may comprise plotting glycan first marker (e.g. lectin) density data for the one or more particles within the sample on a first axis, and plotting the moderated indication of the second marker present for the same one or more particles within the sample on a second axis. This may be particularly advantageous because the combination of glycan first marker (e.g. lectin) data, in combination with other markers, enables improved analysis as the glycan first marker (e.g. lectin) data provides extra dimensions of information beyond conventional markers alone. For example, the indication of the marker (e.g. second marker) present may comprise an indication of lipids present. Graphically plotting the moderated indication of the marker present may then comprise plotting glycan first marker (e.g. lectin) density data for the one or more particles within the sample on the first axis, and plotting the moderated lipid density data for the same one or more particles within the sample on the second axis.

[0764] Whilst the above disclosure discusses using the forward scatter signal to moderate the indication of the marker present based on an indication of size for said one or more particles, the skilled person will understand that this is not limiting, and that indication of size may be determined in other ways.

[0765] Incorporation of size moderation, in particular based on surface area and / or volume normalisation, to basic flow cytometer output may be advantageous universally across many different areas of cell biology, cell cycle, stem cell differentiation, and development, etc. As one example, the method may be advantageous for analysing the cell death programme, for example analysing exit of apoptosis into necrosis. Size moderation as disclosed herein may be particularly effective for cell death analysis because the cell death process contains substantial cell changes, such as when cells reduce in size as apoptotic bodies are formed, and further reduction in size when microvesicles are formed.

[0766] However, the skilled person will understand that substantial size changes are not unique to the cell death programme, and as such the method may also have advantageous applications for other biological analyses.

[0767] The viability marker may comprise, but is not limited to, at least one of:(i) Annexin V, and wherein identifying the first subpopulation of particles based on the corrected indication of the Annexin marker comprises identifying the first subpopulation of particles to be dead by apoptosis; or

[0768] (ii) 7-AAD, and wherein identifying the first subpopulation of particles based on the corrected indication of the 7-AAD marker comprises identifying the first subpopulation of particles to be dead by necrosis.

[0769] The flow cytometer data may further comprise a forward scatter signal, FSC, for each particle, wherein the indication of size is obtained based on the forward scatter signal for each particle, for example as was described in the first aspect of the invention.

[0770] EXAMPLES SECTION

[0771] Senescence detection - M&Ms and Examples 1-4

[0772] Materials and Methods

[0773] Cell culture

[0774] Mesenchymal stem cells derived from human dental pulp (DP-MSCs, Leipzig University) or from bone marrow (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 endothelial cells (HUVEC; PromoCell) were cultured using a ready to use endothelial cell growth medium (C-22010; Promocell). Human fibroblasts (StemNovate) were cultured using a ready to use fibroblast growth medium (C-23025; Promocell). All different cell types were incubated in standard condition (37°C with 5% CO2).

[0775] Surface marker expression - cell identity

[0776] Dental pulp derived MSC were tested for the expression of specific surface markers by flow cytometry, using the MSC phenotyping kit (130-095-198; MiltenyiBiotec). This kit consists of two antibody cocktails: phenotyping (CD73-APC, CD90-FITC, CD105-PE, and CD34 / CD45 / CD14 / CD20-PerCP) as an isotype control cocktail. Briefly, MSCs (5 x 105cells) were suspended in staining buffer consisting of 2mM EDTA and 3% FBS in PBS and incubated for 10 min at 4°C with either phenotype or isotype antibody cocktails. Data was acquired with the BD FACSCanto™ II (BD Biosciences) flow cytometer and analysed using Flowjo software version 10 (Flowjo). A compensation matrix was generated using single antibodies from the kit. Doublets were excluded using FSA vs FSH gating for all samples, before gating on total live cells, negative for 450 viability dye (562247; BD Biosciences). Bone marrow MSC were tested in the same manner but just for the following markers: CD45, CD73, CD90, CD105 and found to be positive for CD73, CD90 and CD105 and negative for CD45 (Table 1), as iscommon for MSC. For the HUVEC characterization we used the following markers: CD31, CD45 and CD146 and found to be positive for CD31 and CD146 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. Table 1. Immunophenotype of HUVEC, fibroblasts, BMMSC and DP-MSC by flow cytometry.

[0777]

[0778] The HUVEC and the fibroblast populations from 3 different donors were poo ed. BMMSC (n=3 individual donors) and DPMSC (n=5 individual donors).

[0779] Senescence induction

[0780] Senescence was induced by exposure of proliferating cells to 200 nM Doxorubicin (Doxo, 5927S; Cell Signalling Technology) dissolved in DMSO for 48 hours. Vehicle control samples were treated with equal concentration of DMSO. The cells were washed with PBS following incubation with either the drug or DMSO for 2 days and then cultured with fresh drug-free culture media for 10 additional days before determining senescence level. All experiments were replicated and performed with appropriate controls.Flow cytometric senescence assessment

[0781] Cells were subjected to flow cytometry analysis for senescence assessment with the simultaneous study of any the following markers; beta-galactosidase activity with DDAO (11540346; Fisher scientific), expression levels of p16 protein (ab209579; Abeam), yH2AX protein (pS139; BD Biosciences), ki-67 protein (561283; BD biosciences) and cell size with forward scatter (FSC). The detailed procedure as well as the concentrations used for the senescence assessment with the above markers has been described in detail at Adeolu et al., 2020.

[0782] SASP secretion

[0783] The secretion of IL8, IL6, and MCP1 proteins in the culture medium was measured using Luminex technology according to the manufacturer’s instructions (R&D Systems). The cells at different stages of senescence were cultured for 2 days in serum-free culture medium (A1033201; ThermoFisher). Then, the culture medium for each condition was collected, and centrifuged at 300x g for 5 min, and that supernatant was stored at -80C until analysis. Data acquisition was performed on a Luminex MAGPIX™ Multiplex Reader (BioRad). Data was normalised to cell number for each condition.

[0784] Lectin staining and flow cytometry analysis

[0785] To assay cells, after washing adhering cells, adherent cells were mildly 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.

[0786] Briefly, MSC (Bone marrow and dental pulp), 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 10min 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 5x105cells 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 secondaryantibody in blocking buffer for further incubation of 30min at 4°C. Finally, cells were washed (300g, 5min) and resuspend in blocking buffer prior analysis with the flow cytometer.

[0787] MSC magnetic isolation and quantification

[0788] Cell number of young or Doxo-treated cells was determined using an automated cell counter (NucleoCounter®NC-3000™, Chemometec) following the manufacturers’ recommendations. After centrifugation (300g, 5 min) the supernatant was removed, and the young cells were stained with CellTrace Violet (CTV; C34571; Thermofisher) in PBS, while the Doxo-treated cells were stained with CellTraceFarRed (C34572; Thermofisher) in PBS for 15min at 37°C. Cells were then washed in culture medium (300g, 5 min), resuspended in blocking buffer and incubated for 10min at room temperature. After a wash with PBS, both young and Doxo-treated cells were mixed in 1:1 ratio, incubated with different lectins previously diluted in blocking buffer for 30min at 4°C. Subsequently, the mixed population was washed in blocking buffer, resuspended in 100pl of cold autoMACS buffer (130-091-221; MiltenyiBiotec) supplemented with 0,5% BSA (A9418; Sigma-Aldrich) and incubated with 10pl affinity microbeads for 15min at 4°C. Cells were washed and resuspended in 600pl BSA in autoMACs buffer and were passed twice through an MS column (130-042-201; MiltenyiBiotec) attached to an OctoMACS separator (130-042-109; MiltenyiBiotec), following company’s recommendations. The mixed population before the magnetic separation, as well as the cells from the flow-through (passed through the magnet) and the bound cells (preserved in the column), were all subject to analysis by flow cytometry.

[0789] Statistical analysis

[0790] Statistical analyses were performed using Prism 8 (Graph Pad) using Student’s t-test or oneway 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.

[0791] Senescence detection - Examples 1-4

[0792] Example 1- Successful drug induction of senescence in endothelial cells (HUVEC) and fibroblasts

[0793] Human primary endothelial cells, HUVEC, and human primary fibroblasts were subjected to doxorubicin (DOXO) treatment, to induce senescence. Flow cytometry analysis of three markers on senescent HUVEC show elevation post DOXO treatment: (1) beta-galactosidase activity as measured by DDAO (Figure 1A, B); (2) p16 protein levels (Figure 1C, D) and (3) cell size as indicated by forward scatter (Figure 1E). Same analysis in DOXO treated fibroblasts demonstrated elevated (1) beta-galactosidase activity (Figure 2A, B) and (2) cellsize (Figure 2C), in comparison to the young, untreated cells. These data show DOXO treatment of HUVEC and fibroblasts induces senescence conforming to accepted markers.

[0794] Example 2 - Confirmation of MSC senescence upon prolonged culture expansion (replicative senescence) and after DOXO treatment

[0795] Human primary MSC isolated from either bone-marrow (BMMSC) or dental pulp (DP-MSC), were either culture expanded for various passages or treated with DOXO for senescence induction and were subjected to senescence assessment (Figure 3). Flow cytometric analysis of four markers of senescence demonstrated an increase in betagalactosidase activity (Figure 3A) as well as in p16 (Figure 3B) and yH2AX (Figure 3C) protein levels but a decrease in ki-67 (Figure 3D) protein levels in both replicative- and induced-senescence. To further confirm the establishment of senescence upon replicative- and induced-senescence of MSC, we studied the secretion levels of IL-6 (Figure 4A), IL-8 (Figure 4B) and IGFBP-2 (Figure 4C), which are all part of the senescence-associated secretory phenotype (SASP), with Luminex technology. These secreted proteins all show significant elevation upon prolonged expansion and DOXO treatment (Figure 4). All the above changes are in accordance with the literature and clearly demonstrate senescence establishment (Adeolu et al., 2020).

[0796] Example 3 - Cell surface binding by lectins differentiates senescent cells from young cells.

[0797] Having established confidence in the drug-induced and replicative senescence models, the inventors applied a panel of lectins targeting specific terminal carbohydrate residues by flow cytometry to detect cell surface binding differences between senescent and young cells. The inventors demonstrate that for three different cell types, HUVEC, fibroblasts and MSC. One lectin, UEA-I, while showing weak binding for BMMSC and fibroblasts, show strong binding on young HUVEC cells (Figure 5 and 6). By contrast to other lectins, UEA-I exhibit reduced binding upon DOXO-treated HUVECs (Figure 5 and 6). Both AAL and UEA-I bind fucose residues, but differ in the linkage between fucose and the underlying carbohydrate residue. It is interesting, how drastically different UEA-I binding is between the cell types while AAL is binding consistently. It is very significant that UEA-I declines rather than increases upon senescence in HUVEC cells.

[0798] Replicative senescence for DPMSC induces a similar profile of lectin binding changes, where MAL-II, SNA, GNA, UEA-I, STL and SJAall exhibit increased binding with increased passages(Figure 7 and 8). UEA-I binds weakly but also increases upon DOXO treatment on DPMSCs (Figure 7 and 8). DOXO treatment of DPMSC in comparison produces the same lectin changes, apart from AAL binding which consistently goes down (Figure 9 and 10). Finally, DOXO treatment of DPMSC increases NPL binding (Figure 9 and 10).

[0799] These data present a consistent trend of increased lectin binding in MAL-II, SNA, NPL, GNA, AAL, STL, SJAand PNAupon replicative senescence. In induced senescence, the binding of all the above lectins is consistent, except AAL which significantly drops after DOXO treatment in DPMSC. UEA-I is showing weak binding to fibroblasts and BMMSCs but changes upon senescence for DPMSC and HUVECs.

[0800] Example 4 - Purification of young or senescent cells based on their lectin content Following the study of lectin binding in different cell types upon senescence, the inventors selected the following five lectins to attempt to enrich young cells from a 1 :1 ratio of young and DOXO treated cells: AAL, NPL, MAL-II, GNA and SNA (Figure 11). These lectins were used independently or as a mixture to isolate cells using magnetic affinity beads (Figure 11). The inventors achieved enrichment of young cells to 150% compared to the old cells in the flow-through population for HUVEC (Figure 11 A). Over 300% enrichment was seen for fibroblasts (Figure 11 B) and 200% of BMMSC (Figure 11 C). These data show that lectins independently or as a mixture can enrich young cells from a mixed population.

[0801] Early Apoptosis detection - Materials and Methods and Examples 5-7

[0802] Materials and Methods

[0803] Cell culture

[0804] 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).Surface marker expression - cell identity

[0805] 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 2), as is common for MSC. For the HUVEC characterization we used the following markers: CD31 , CD45 and CD146 and found to be positive for CD31 and CD146 and negative for CD45 (Table 2), as is common for endothelial cells. Finally, the human fibroblasts were tested positive for the CD90 and negative for CD45 surface markers (Table 2), as is common for fibroblasts.

[0806] Table 2. Immunophenotype of BMMSC, HUVEC and fibroblasts by flow cytometry. The

[0807]

[0808] HUVEC and the fibroblast populations from 3 different donors were pooled. BMMSC (n=1 individual donor).

[0809] Extrinsic and intrinsic apoptosis induction

[0810] Extrinsic apoptosis was induced on MSC by anti-FAS monoclonal antibody (aFAS; CH11; Merck) treatment followed by 6 hours incubation of cells for sufficient apoptosis induction. Untreated (control) MSC were also included.

[0811] Intrinsic apoptosis was induced on MSC, fibroblasts and HUVECs by treatment of with 1 M 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).

[0812] Lectin staining / AnnV staining and flow cytometry analysis

[0813] 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 10min 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 5x105cells 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.

[0814] Statistical analysis

[0815] Statistical analyses were performed using Prism 9 (Graph Pad) using Student’s t-test or oneway 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.

[0816] Early Apoptosis detection - Examples 5-7

[0817] 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 negative populations of primary cells (i.e., bone marrow derived MSC, fibroblasts and HUVEC).

[0818] 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 GNAand 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 GIcNAc terminating glycans. Changes to MAL-II binding, indicating exposure of a-2,3 sialic acids, was also seen.Example 5 - Intrinsic apoptosis induces early-staqe cell surface qlycan fluctuations in fibroblasts before phosphatidyl serine exposure

[0819] 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 binding is not fully activated (Figure 1B). 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 1 A). 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 binding has not been fully stimulated by staurosporine (Figure 1B). 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).

[0820] Example 6 - Bone marrow derived mesenchymal stem cells (BMMSC) mirror fibroblasts in exhibiting early plant lectin binding divergence during intrinsic and extrinsic apoptosis

[0821] 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 SJAis 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 pre-commitment phase of apoptosis (Figure 2C).

[0822] 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).Example 6.1 -Apoptotic bone marrow derived mesenchymal stem cells (BMMSC) BMMSCs were treated with staurosporine, and stained with a lectin (having a detection moiety) and annexin A at 15 minutes or 1 hour post treatment. An untreated control was likewise stained. Separate experiments were stained with separate lectins, namely STL, SNA, MAL, GNA and AAL. The stained samples were analysed by flow cytometry, to generate 2D contour plots (Y axis = MFI for lectin; X axis = MFI for annexin A). The contour plots were overlaid with a grid matrix having nine segments, thus outlining nine dimensional spaces. The position of contours in dimensional space could thus be analysed overtime.

[0823] Example 7 - HUVEC exhibiting qlycan changes during early intrinsic apoptosis

[0824] 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. NPLand 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).

[0825] 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.

[0826] Detecting Apoptosis substages across entire programme - Examples 8-12

[0827] Example 8 - Enhanced resolution of apoptotic substages through combined Annexin V and lectin analysis

[0828] Apoptosis is traditionally characterised using flow cytometry by detecting the externalisation of phosphatidylserine (PS) through Annexin V staining. This one-dimensional (1 D) approach, while effective for broadly identifying apoptotic cells, fails to capture the complexity of the apoptotic process. Our initial 1 D analysis of Annexin V staining across an 18-hour time course,following staurosporine-induced apoptosis, revealed an increasing PS-positive population, indicative of apoptosis progression (Figure 17A(top)). However, this method was limited in resolving the nuanced transitions and subpopulations that emerge during apoptosis.

[0829] To address this limitation, we employed a two-dimensional (2D) analysis that combined Annexin staining with GNA(Galanthus nivalis agglutinin) lectin detection, which is sensitive to mannose terminations. This approach significantly enhanced the resolution of apoptotic subpopulations, allowing us to identify six distinct subpopulations that were not discernible through 1D analysis alone (Figure 17A(bottom)). These subpopulations reflect a structured and dynamic Lectomic programme that parallels the intricate internal molecular changes during apoptosis, providing a more detailed understanding of the process.

[0830] Further analysis with additional lectins, including SNA (Sambucus nigra agglutinin) and AAL (Aleuria aurantia lectin), at critical 12-hourand 18-hour time points, reinforced the utility of 2D analysis. These lectins revealed that glycosylation changes are particularly crucial at advanced stages of apoptosis, distinguishing subpopulations 5 and 6, where a resurgence of glycan expression occurs on the cell surface (Figure 17B). This late-stage re-glycosylation appears to play a critical role in the apoptotic process, particularly at the high PS exposure stages.

[0831] By normalising lectin binding against cell surface area using the FSC-A parameter, we derived a more accurate representation of PS exposure, correcting for cell size differences that can confound traditional signal-based interpretations. This density-normalised approach provided clearer insights into the true state of PS exposure, revealing that previously observed slight increases in Annexin signals were misinterpreted due to concurrent cell size changes (Figure 17C).

[0832] To facilitate the interpretation of these complex data, we introduced a novel "sugar wheel" representation, which visually summarises Lectomic data by categorising key glycan structures into a wheel format. Each sector of the wheel corresponds to a specific glycan, such as sialic acid or mannose, with subdivisions representing linkage-specific structures. This innovative visualisation method offers an intuitive and comprehensive overview of glycan dynamics during apoptosis, setting a new standard for Lectomic data representation.

[0833] Example 9 - Two-dimensional lectin-PS analysis reveal novel substaqes of apoptosis Apoptosis is generally defined using flow cytometry, after gating out dead cells, which have permeable membranes, the phosphatidyl serine (PS) positive population, using Annexin V. We upgraded this one-dimensional analysis of apoptosis by combining Lectin and PS detection to reveal previously unidentified substages of apoptosis. This disclosure exploresthe apoptosis process holistically by examining all substages at once. We specifically highlight five main discoveries at key transition phases of apoptosis:

[0834] 1. Cell to apoptotic body transition: wholesale deglycosylation

[0835] 2. Three stage of apoptotic body maturation: re-glycosylation

[0836] 3. Exit of apoptosis to necrosis: PS++ / Glycan rich apoptotic bodies

[0837] 4. Pre-PS stage: deglycosylation

[0838] 5. Distinction of membrane blebs which are PS negative and a subfamily of PS+ vesicles with dynamic Lectomics

[0839] It is well accepted that apoptosis produces apoptotic bodies of size 0.5pm~2.0pm. One dimensional lipid-based analysis using Annexin-V, produce some visible separation of the ‘mother cell’ (M pop) and the arising ‘apoptotic bodies’ (Figure 17a, i: histogram of AnV for each time point, dotplot of FSC vs AnV and Dot plot of SSC vs AnV). Lectomic-lipid combinational 2D analysis creates dynamic separation of the mother (M) pop from the apoptotic bodies, in particular, we can further distinguish the M’ and M” populations which are cells with increasing PS exposure, rather than PS positive vesicles. (Figure 17b- hour 7-12h). Across the 18 hours of induction of intrinsic apoptosis by staurosporine, we observe clear changes from the cellular population to apoptotic bodies, which through conventional onedimensional PS only analysis cannot be clearly resolved. Using physical parameters of standard flow cytometers, forward scatter (FSC) and side scatter (SSC) there is improved separation of cells from apoptotic bodies. However, when compared to 2D Lectin-PS analysis the superior separation of M from apoptotic bodies at hour 7, the first point of apoptotic body appearance, is clear. Significant overlap of M” and the first apoptotic body pop 4 is still seen when PS is plotted with physical parameters FSC or SSC (Figure 17C).

[0840] Following the above observation of clear distinction between cell from the first apoptotic body subpopulation, pop 4, it is clear that significant de-glycosylation has taken place in this first stage of transition.

[0841] Investigating further the PS positive populations using one dimensional analysis of Annexin V, we only observe two populations of apoptotic bodies with varying levels of PS exposure. Interestingly 2D lectomics-lipid analysis reveal three distinct states of apoptotic bodies (pop 4-6), with the last stage (pop 6) being highly glycosylated. This final population of pop 6 completely overlaps with pop 5 in traditional one-dimensional analysis.Example 10 - Sugar density map unifies understanding of cell and apoptotic transition Cell size changes greatly during the birth of apoptotic bodies. To scientifically quantify the changes in surface expression of glycan classes and to be able to compare between cells and apoptotic bodies, we sought to normalise against surface area. Since the FSC-H parameter of the flow cytometer represents the diameter of the passing cell, the squaring of FSC-H therefore is a proportional representation of the surface area of the cell. Normalising against the square of FSC-H we derive Lectome density. This quantification method unifies reporting of Lectome density across all cells and vesicles with varying cell size.

[0842] delta Lectin binding

[0843] Lectin Density = - FSC - A

[0844] delta Lectin binding = Lectin X gMFl — secondary only gMFI Lectomic signatures are represented in sugar wheels (Fig 18) as well as dot plot format. With surface area normalisation, we can for the first time make a statement about the overall desialylation and deglycosylation during the birth of apoptotic bodies. However, while it is mechanistically easier to explain the loss of glycans in the formation of apoptotic bodies (pop 4), a conundrum arises when explaining the dramatic increase of glycans in pop 5-pop 6 transition at the end of the apoptotic process from 12-18 hours. This phenomenon is especially puzzling when considering the apoptotic body lacks functional ER and golgi. Our duplex lectomic panel, combining plant lectins and GNAand in addition to Annexin V, show that over 90% (Figure 19) of the pop 6 cells simultaneously express all glycans predicted by these lectin binding.

[0845] Example 10b - Lectomic signature of the entire apoptosis process

[0846] Using the technologies described, we are able to uncover the first Lectomic signature map of the entire apoptosis process (Figure 18) - the data comes from MSC cells of the type mentioned in the Examples section. By applying density normalisation to four different particle populations: mother cell, apoptotic bodies, necrotic bodies and vesicles, we were able to, for the first time, compare quantitatively across these different particles of highly variable sizes. As a non-apoptotic cell begins commitment towards apoptosis, we observe that the cell size remains relatively stable as PS gradually increases during the first 7 hours. There is a dramatic decline in cell size at around 7 hours that marks the beginning for the formation of first apoptotic bodies. Three distinctive apoptotic body types emerge from hours 7 to 18 hours, marked by a wide range of PS and Lectin binding. Highly contrasting Lectomic signatures are found between these apoptotic bodies. Entry into necrosis, which traditionally has been simplyconsidered as a single population movement is in fact revealed to take two routes: one from Pop 5 to Pop 8, characterised by an overall suppressed glycan display and the other from Pop 6 to Pop 7, characterised by highly elevated glycan displays. Multiple subpopulations of vesicles are definable through 2D analysis of PS and lectins. Two of these subpopulations are chosen as representatives: Pop 9 stemming from Pop 6 and Pop 10 stemming from the mother population, M pop. Together, this technology creates a “defined kit” of a new map of apoptosis programme, highlighting novel pathways and permitting scientific quantitative analysis.

[0847] Calculations:

[0848] Following gating as shown in the example above, lectin and FSC-Adata is extracted from the .fsc file. Density of Lectin binding for each population is calculated, then subsequently expressed as a percentage compared to the Mother population, pop M, for which each lectin: AAL (fucose binder), GNA (mannose binder) and SNA (alpha-2,6 sialic acid binder) is expressed as relative lectin density. In short, these three classes of glycan terminations, fucose, mannose and sialic acid, is expressed as a percentage change as apoptosis progresses from the healthy cell.

[0849] The lectomics profiles of Figure 18 are illustrated in tabular form below:

[0850]

[0851] Example 11 - Apoptosis to Necrosis transition revealed: immunological relationship Exit of apoptosis into necrosis lies at the heart of a deep immunological question: how is the immune system triggered and regulated. Polly Matzinger first proposed the danger hypothesis that regards externalisation of intracellular antigens as the main source of danger signals. The raison d’etre of apoptosis therefore is primarily in maintaining membrane integrity andpreserving the lack of immune initiation. However, as apoptosis transitions to necrosis, there is increased membrane permeability. Lectomic analysis for the first time reveals which of the sub-populations is the exiting population of apoptosis (Figure 21). Necrotic exit appears in pop 4. However, this population mostly retain membrane integrity from 12h to 18h. The main two populations of necrotic exit are pop 5 and the re-glycosylated pop 6. At 12h there is proportionally more pop 6 exit compared to pop 5, however, this reaches equality at 18h.

[0852] Example 12 - Lectomic analysis separates membrane blebs from PS+ vesicles.

[0853] Membrane blebbing is a hallmark of this early phase of apoptosis and two types of membrane blebs, which are vesicles, are known. One is an expression of ER material and derive from ER membrane while the other is nuclear material and include histones and derive from the plasma membrane (PM). These blebs, especially the PM type would be decreasing cell surface glycans as such glycosylation is carried away as the blebs are formed and released from the cell surface. This provides an explanation for the decrease in the majority of the glycans observed in the first few hours of the M pop. Inhibition of the membrane blebbing decelerates this loss of glycans observed.

[0854] We explored the area of the flow cytometer plot that is outside of the conventional cell and apoptotic body gate, which we named as the ‘vesicle’ gate. We observed specific increase in proportion of this ‘vesicle’ gate from 7h - 18h. This is the period where in one dimensional lectin plots we can observe specific sugar profiles, e.g. SNA’s sharp rise at hour 12, gradual increase of mannose (GNA) and steady expression of fucose (AAL). However, two-dimensional lectin-PS expression highlight to distinct tracks of vesicle formation. One track is marked by no PS expression while the other is very high in PS exposure. We observe that the PS++ vesicle population emergence is highly correlated with high level expression of all lectins *(GNA / AAL / SNA) and their emergence is restricted to hour 12-18, whereas the PS negative vesicle population show distinct profiles with a decline after hour 1-7. This unique way of combining lectin and PS analysis shows that fucose terminating vesicles in fact come from two sources: PS negative (before hour 7) and PS positive (from hour 12 onwards). We explored the source of the PS negative and positive vesicles and concluded (exp) that PS negative vesicles represent membrane blebs from the mother cell while the PS positive vesicles are coming uniquely from the de novo Pop 6 which carries high levels of glycosylation. (Figure 22)

[0855] By using the algorithm for sugar density normalisation and combining lectin and PS quantification in 2D flow analysis, we explored vesicle and apoptotic body sugar display in conjunction with mother cell populations and necrotic transit populations. These technologicaladvances allowed study of all ten stages of apoptosis (Figure 18) simultaneously and permit quantitative comparison of glycan expression across the apoptosis process.

[0856] Example 13 - Further examples of 2D analysis

[0857] [Experimental design] In this experiment, unhealthy and healthy human T cells from peripheral blood monocyte cells (PBMC) are compared to evaluate death process during drug treatment. Staurosporine is known to induce intrinsic apoptosis and was applied over 16 hour time course with untreated and DMSO vehicle only controls are included. When applying traditional analysis using the market leader, Annexin V, that binds to lipid, phosphatidyl serine (PS) on the cell surface, we can create the 1 dimensional analysis (Fig 23 top panel). All time points of staurosporine treatment including the controls are overlaid to create a comprehensive overview. So in this example 4 time points were overlaid. Therefore the overlay is a ‘composite view’ over time over several individual snap shots of the population taken at said times reporting level of PS.

[0858] The bottom panel in figure 23 represents a 2 dimensional view of the same samples (time condensed snap shots).

[0859] [2D advantage] Limited information relating to the changes in the cell is visible in 1 dimensional analysis (Top panel) compared to 2-dimensional analysis (bottom panel) of Annexin-V plotted against a panel of plant lectins (or any terminal carbohydrate recognising reagent), specifically chosen to cover the range of possible terminal carbohydrate residues. In 1 dimensional analysis, it is only possible to discern two major regions of interest, PS negative and positive, thus producing a somewhat binary output. This can be used to crudely judge what percentage of cells are past the apoptosis checkpoint of PS moving to the cell surface after caspase 3 activation). By contrast, vivid sub-population details and resolution is possible within the 2 dimensional analysis of the same data due to the combination of the single cell analysis of the single cells in the population and the orthogonality and positive / negative mapping relationships between sugar and lipid during the death process. Important to note that the embodiment has increasing utility as more lectins are simultaneously compared in the samples against the same annexin signal of overlain condensed signals. This allows a more nuanced view of the interrelationship of sugar terminal carbohydrate display on the cell surface (in this case) as apoptosis progresses. The investigator can then interpret the overall data displayed through the multiple overlain views of condensed snap shots at individual timepoints to identify differences in terminal carbohydrate displays at the chosen particle boundary (in this case the cell surfaces). The experimental design can be changed to incorporate additional markers (no pre PS markers but may wish to have a different marker for another experimental design) that pinpoint an alternative timeframe window of investigation, for example pre-ps exposure in thisexample to identify pre-PS exposure apoptotic changes in terminal sugar presentation on proteins on the cell surface.

[0860] Additionally, investigators can interrogate the raw uncondensed (non overlapped) time series data for more nuanced changes) - in other words condensing is useful but optional.

[0861] [Time I synchronisation] Regarding a cell line, which was originally derived from a single cell from a patient, its behaviour during this experiment is expected to be somewhat synchronised compared to healthy human T cells, which may contain multiple compartments such as effector, memory, effector memory, naive, regulatory, etc. Furthermore, it is also important to note that MSC cells, used in specific apoptosis example later is also more heterogeneous than the synchronised cells and thus we spot the multiple populations without collapsing timepoints. The technology of time compression, or overlaying of multiple timepoints together, overcomes the loss of sub-population relationship information in synchronised cells and accentuates the orthogonality / mapping qualities found in 2D plots.

[0862] [Agnosticism] The agnostic kit, which can have broad or specific sugar termination coverage, is only presenting resolution and subpopulation relationships, rather than stating specifically what each population is. This is a fundamental attribute of the innovation. The subpopulation information derived (through further interpretation and experimentation) can help users to identify for example drug specific effects, healthy / unhealthy differences, find better targets for treatment, select more suitable or efficacious drugs, etc. The subpopulation information also helps users to design separation experiments to then apply multi-omics and imaging analysis to better understand the new subpopulation that was once hidden in 1 dimensional analysis. The agnosticism in this technology stops at the point of displaying subpopulations and leaves room for users to further explore the features identified, without dictating to the user how to interpret.

[0863] Importantly, using the same agnostic principle, the investigator can modify the experimental protocol to for example synchronise cell populations that in this case will allow time based interpretation that could be used with the technique of overlaying the resultant data in a overlaid condensed view, or to interpret each individual time snap shot of data as time progresses.

[0864] Example 14 - Cell cycle monitoring

[0865] T cells from healthy donors are stimulated to activate using CD28 / CD3 cross-linking. Cell trace (CTFR, Cell trace far red dye) is applied to work out number of division the T cell has completed. Edu / DAPI is applied to work out phase of the cycle its currently in. Combining these two dimensions of information, the CTFR + EDU / DAPI combo defines a coordinate of Tcell cycle / phase. Lectin panel is applied after 4 days of incubation / stimulation. T cells are stained for CD8 and CD4 expression and catalogued based on high and dim expressions of these markers.

[0866] Below is a detailed example of a CD8 dim T cell, with all lectin binding density data compared relative to the division 0 (not divide) CD8 dim T cell, in its G1 phase of the cycle.

[0867] This first table is the percentage of Lectin density compared to G1 of Division 0 of CD8 Dim. Here N4 is the donor code. Px is the number of division that CD8 Dim cell has undertaken in four days. G1 / S1 / MidS / S2 / G2M are divisions of the cell cycle. S1 is early S phase, MidS is middle of the S phase and S2 is the late S phase. G2M is the combinational grouping of G2 and M phases.

[0868] Styphnolobium japonicum agglutinin (SJA), in particular here SJA-I is targeting termination of a D-galactose / N-acetyl-D-galactosamine, i.e. terminal Gal or terminal GalNAc.

[0869] Example 15 - T cell differentiation

[0870] This example is a defined used method for looking at T cell differentiation. It illustrates one application of the methodology in cataloguing cell differentiation from stem cells and to aid discovery of subpopulations, which is very valuable in the field of immunology and other disciplines. One thing to note is that this example utilises CD4 and CD8 which are protein markers in conjunction with a lectin panel. Thus it illustrates an example where two non-glycan markers are pitted against lectins.

[0871] Lymphocytes include T cells, B cells and NK cells. These cells are known to have a diversity of subsets, classically defined protein based markers such as CD4, CD8, CD25, etc. In this example, we are taking the classical biomarking system of T cells, further in two ways. The first is simply assigning Lectomic signatures for different major categories of T cells based on CD4 and CD8 protein expression. A summary of these sugar signatures are displayed in sugar wheel format and annotated in the differentiation map (Figure 26) of the lymphoid arm of haematopoietic stem cell differentiation programme.

[0872] The second is to go a step beyond simple assigning of lectomic signatures, which utilise the average signal of each lectin channel. By exploring the distribution of expression of each lectin in the panel, at a single cell level, we can spot subpopulation details that is not revealed by the sugar wheel (see Figure 27). For example MAL-II in CD4dim T cells show two peaks while it’s a single peak in CD4hi population. This is significant, because it implies orthogonality between MAL-II and CD4, i.e. at a particular CD4 expression - the dim level, MAL-II is able todiscern two subpopulations while CD4 only sees one. This concept can be applied ad infinitum, to incorporate further levels of subpopulation analysis in all cell types.

[0873] This example illustrates the utility of the lectomic approach to characterise and to aid discovery of novel interesting subpopulations in cell differentiation pathways. In this particular example, two markers, CD4 and CD8, which both are protein targets is measured in parallel to the lectin panel.

[0874] The lectomics profiles forTcell differentiation as seen in Figure 27 are outlined in tabular form below:

[0875]

[0876] Examples 15-17 - size moderation

[0877] The following examples illustrate the range of changes size or density moderated data produce when compared to “raw”, unmoderated data. In light of these changes, it becomes clear that historical cytometry data, which lack this key compensation step, need to be reexamined. This re-examination has the potential to impact scientific definitions, key findings and accepted dogma, to drug approval decisions.

[0878] Example 15 - Necrosis and Apoptosis analysis

[0879] Fig. 31 A shows an example plot of flow cytometer data wherein “raw” data relating to marker 7AAD is shown on the y-axis, and “raw” data relating to marker Annexin is shown on the x-axis. It is well known that there is an ambiguous gap between live and dead subpopulations when stained with well characterized viability dyes such as 7AAD, where users are unsure whether cell are dead or alive.

[0880] A healthy cell population is indicated by plot 402, whereas a dead cell population is indicated by plot 404. Classical definitions of raw 7AAD boundaries define a “live” zone (bottom right) and “dead” zone (top left) from untreated MSC samples. A sample cell population is represented as plot 406, in this case a MSC 12-hour drug treated sample.

[0881] As shown in Fig. 31 A, many cells are observed within the ambiguous 7AAD gap 408 where it is unclear whether cells are “live” or “dead”. It is therefore difficult to distinguish live cells in the sample 406 from dead ones, not least because there is an ambiguous gap 408 between the control alive population 402 and dead population 404.

[0882] Fig. 31 B shows an example plot of the same flow cytometer data wherein density-moderated data relating to marker 7AAD is shown on the y-axis, and “raw” data relating to marker Annexin is shown on the x-axis. In contrast to Fig. 31 A, a clear-cut discrimination threshold emerges for identifying live or dead cells, as indicated by line 410. As such, by applying the density 7AAD axis, an immediate collapse of the ambiguous zone is observed, thus making interpretation and identification of live I dead cells much simpler. Density moderation of flow cytometer data can therefore be shown to facilitate new and improved necrosis boundary corrections.

[0883] Fig. 31 C shows an example plot of flow cytometer data wherein “raw” data relating to marker 7AAD is shown on the y-axis, and density-moderated data relating to marker Annexin is shown on the x-axis. Fig. 31 D shows another example plot of the same flow cytometer data wherein density-moderated data relating to marker 7AAD is shown on the y-axis, and densitymoderated data relating to marker Annexin is shown on the x-axis. When Annexin data ismoderated according to density, improvements in clarity of subpopulations separated by Annexin density boundaries is observed compared to Annexin raw boundaries. By way of illustration, a first sub-population boundary is indicated by arrow 412, and a second subpopulation boundary is indicated by arrow 414. In both instances, the boundary becomes more defined when Annexin is moderated based on cell size. Density moderation of flow cytometer data can therefore be shown to facilitate new and improved apoptosis boundary corrections, and assist in identifying cell subpopulations.

[0884] It is indeed surprising that, using vDEN correction of 7AAD and sDEN correction for Annexin V, without any additional staining, the same data transforms in clarity and utility.

[0885] Since this gating step is universal and one of the first gates applied to flow cytometry analysis, the impact of a small shift in these gates are monumental, especially considering the long history of flow cytometry.

[0886] While the mathematics of vDEN and sDEN may be perceived as relatively straightforward, forty years of omission of this crucial correction step argues for non-obviousness in its discovery.

[0887] Example 16 - Cell vesicle analysis

[0888] Since vesicles are approximately one tenth the size of a normal cell, the impact of density correction becomes great. This sensitivity to density is illustrated by Figs. 32Aand 32B which compare traditional raw fluorescence-based axis plotting (Fig. 32A) versus new density-based axis plotting (Fig. 32B). The density-based moderation of the present invention facilitates a new, sensible, and quantifiable comparison of the relative surface expression levels of markers between cells and vesicles.

[0889] In the example shown in Figs. 32Aand 32B, cells are treated for 4 hours with a drug which is expected to induce much vesicle formation during apoptosis.

[0890] Fig. 32A shows an example plot of flow cytometer data wherein “raw” data relating to marker Annexin is shown on the x-axis, and “raw” data relating to lectin marker NPL (Narcissus Pseudonarcissus (Daffodil) Lectin) is shown on the y-axis. The plot illustrates marker expression of vesicles 502 and of the cell population 504.

[0891] Fig. 32B shows an example plot of the same flow cytometer data as Fig. 32A, wherein surface density moderated (sDen) data relating to marker Annexin is shown on the x-axis, and surface density moderated (sDen) data relating to lectin marker NPL is shown on the y-axis. The plot illustrates marker expression of vesicles 502 and of the cell population 504.Comparing the raw plot of Fig. 32Awith the density-moderated plot of Fig. 32B, a dramatic shift is observed that completely changes the outlook on marker expression levels on the vesicles 502 versus the cells 504. While are using only sugars and lipid markers, the same analogy extends to all classes of molecules, including for example DNA and protein.

[0892] Example 17 - Apoptotic body analysis

[0893] Another example which highlights the impact of density moderation of the present invention is shown in Fig. 33. In particular, the density moderated data reveals the appearance of a new subpopulation that was obscured completely by raw fluorescence-based analysis methods. The plots shown in column 602 plot flow cytometer data for a first population of cells, Cell Type 1, wherein “raw” data relating to marker Annexin is shown on the x-axis, and “raw” data relating to various lectin surface markers (LSM) is shown on the y-axis. The plots illustrate marker expression for vehicle control 610, Cell Type 1 cells after an 8-hour drug treatment time (ST) 612, and Cell Type 1 cells after a 16-hour drug treatment time (ST) 614.

[0894] The plots shown in column 604 plot the same flow cytometer data for the same population of Cell Type 1 cells wherein density-moderated data relating to the volumetric density (vDen) of marker Annexin is shown on the x-axis, and the lectin surface density (sDEN) data relating to the various lectin markers is shown on the y-axis. As for column 602, the plots illustrate marker expression for vehicle control 610, 8-hour drug treatment time (DT) 612, and 16-hour drug treatment time (DT) 614.

[0895] The plots shown in column 606 plot flow cytometer data for a population of a second cell type, Cell Type 2, wherein “raw” data relating to marker Annexin is shown on the x-axis, and “raw” data relating to the various lectin markers is shown on the y-axis. The plots illustrate marker expression for vehicle control 616, the Cell Type 2 cells after an 8-hour drug treatment time (DT) 618, and the Cell Type 2 cells after a 16-hour drug treatment time (DT) 620.

[0896] The plots shown in column 608 plot the same flow cytometer data for the same population of Cell Type 2cells wherein density-moderated data relating to the volumetric density (vDen) of marker Annexin is shown on the x-axis, and the lectin surface density (sDEN) data relating to the various lectin markers is shown on the y-axis. As for column 606, the plots illustrate marker expression for vehicle 616, 8-hour drug treatment time (DT) 618, and 16-hour drug treatment time (DT) 620.

[0897] Anew subpopulation is identified specific to Cell Type 2 cells, but missing in Cell Type 1 cells. Most prominently, the appearance of third apoptotic body population in the 8h and 16h drug treatment time points in Cell Type 2 specifically is observed. This was previously obscured because of heterogeneity of cell size within the traditionally defined subpopulations createdby the erroneous fluorescence-based method. It is unthinkable how much of the last forty years of biological research contain errors like this. The surprising and non-obvious nature of this invention is clear it is impact and the long decades of omission. Indeed, the foundational relationships between lectin and marker can completely change when density is applied. Further to Fig. 33, Fig. 34 illustrates data obtained from subsequent testing that illustrates that the new population is not necrotic by using sDEN for a viability dye. Line 702 indicates the start of the new population in all density plots.

[0898] Whilst the description above discloses the use of the forward scatter signal (FSC) as an approximation for the cross-sectional area of a cell, biological body, or other particle, to utilise for surface density moderation (sDEN) and / or volume density moderation (vDEN), the skilled person will understand that the other approaches may be used for moderating flow cytometry data according to size, including for surface density moderation and / or volume density moderation.

[0899] Fig. 35 shows a flow diagram of an example method to this effect. Firstly, the method comprises obtaining flow cytometer data for a biological sample (810). The flow cytometer data comprises an indication of the presence of a marker within the sample. The flow cytometer data may be obtained at a processor 204 from a flow cytometer 202. The indication of the presence of a marker within the sample may comprise a signal indicative of fluorescence for one or more cells and / or particles (such as vesicles, cell bodies, or other particles) within the sample, wherein the sample has been marked with a fluorescent stain configured to bind to a biological molecule of the one or more cells and / or particles.

[0900] The method also comprises obtaining an indication of size of the one or more cells and / or particles (such as vesicles, cell bodies, or other particles) within the sample (820). Said indication of size may be received by the processor 204. As discussed above, the indication of size may be obtained based on the FSC signal in some examples, however this is not intended to be limiting. For example, in other examples, the indication of size may be obtained as a result of image analysis, or by other methods such as based on electrical impedance of said one or more cells and / or particles within the same.

[0901] The method then comprises moderating the indication of the marker present within the sample based on an indication of size of the cell or particle (830). This step may be performed by processor 204. The term “moderation” is used to refer to a normalisation step which intends to correct the impact of size on the flow cytometer signal relating to the expression of markers. An indication of size preferably relates to an indication of surface area and / or volume, however the indication of size may also relate to a parameter from which said surface area or volumecan be approximated, for example such as radius, diameter, circumference, cross sectional area, etc.

[0902] As discussed above, two primary types of moderation are provided: (i) surface density normalisation (sDEN), and (ii) volumetric density normalisation (vDEN).

[0903] Moderation may also be applied on an individual cell I particle basis, or on a population basis, as discussed above.

[0904] Byway of example, Fig. 36 illustrates an alternative method wherein an approximation of the area of a cell or other particle (including but not lim...

Claims

CLAIMS1. A method of characterising the state of a particle comprising a cell membrane, the method comprising:• determining the presence of a glycan first marker on a surface of the particle;• determining the presence of a second marker associated with the particle; and characterising the state of the particle on the basis of these determinations.

2. A method of characterising the state of a population of particles comprising a cell membrane, the method comprising:• determining the presence of a glycan first marker on a surface of particles within a population;• determining the presence of a second marker associated with particles within a population; andcharacterising the state of the population of particles on the basis of these determinations.

3. A method according to claim 2, wherein the presence of the first and second marker is determined in respect of particles within the same population.

4. A method of characterising the state of a first and / or second particle comprising a cell membrane, the method comprising:• determining the presence of a glycan first marker on a surface of a first particle; • determining the presence of a second marker associated with the first particle; and • determining the presence of the glycan first marker on a surface of a second particle;• determining the presence of the second marker associated with the second particle;and• comparing the results achieved in respect of the first and second markers in the first and second particles, andcharacterising the state of the first and / or second particle on the basis of these determinations.

5. A method of characterising the state of a first and / or second population of particles comprising a cell membrane, the method comprising:• determining the presence of a glycan first marker on a surface of particles within a first population;• determining the presence of a second marker associated with particles within the first population; and• determining the presence of the glycan first marker on a surface of particles associated with a second population;• determining the presence of the second marker associated with particles of the second population; and• comparing the results achieved in respect of the first and second markers in the first and second populations of particles, andcharacterising the state of the first and / or second population of particles on the basis of these determinations.

6. A method according to any preceding claim, wherein the presence of the first glycan marker is determined on the external surface of a particle, or of particles in a population.

7. A method according to any preceding claim, further determining the presence of a third, or subsequent marker, and characterising the state of the particle, or population of particles, on the basis of one or more comparisons between the determinations in respect of the first, second, third (and optionally subsequent) markers.

8. A method according to claim 7, wherein the third marker (and optionally any subsequent marker or markers) is a glycan marker, or a non-glycan marker.

9. A method according to any preceding claim, comprising determining the presence of a single glycan marker and a single non-glycan marker.

10. A method according to any of claims 1-8, comprising determining the presence of a plurality of glycan markers and a single non-glycan marker.

11. A method according to any of claims 1-8, comprising determining the presence of a single glycan marker and a plurality of non-glycan markers.

12. A method according to any of claims 1-8, comprising determining the presence of a plurality of glycan markers and a plurality of non-glycan markers.

13. A method according to any of claims 1 to 3, further comprising determining the presence of markers in respect of a second particle or population of particles.

14. A method according to any of claims 4 to 13, further comprising determining the presence of markers in respect of a third, or subsequent, particle or population of particles, and characterising the state of these particles, or population of particles, on the basis of one or more comparisons between the determinations in respect of the requisite markers.

15. A method according to any preceding claim, wherein one or more determination is undertaken in respect of a particle, and one or more determination is undertaken in respect of a population of particles, and the particle and / or population of particles are characterised on the basis of these determinations.

16. A method according to any of claims 1, 6-13 or 15, comprising further steps of:• determining the presence of the glycan first marker on the external surface of a second particle;• determining the presence of the second marker associated with the second particle;and• comparing the results achieved in respect of the first and second markers in the first and second particles, andcharacterising the state of the first and / or second particle on the basis of these determinations.

17. A method according to claim 2, 3, 6-13 or 15, comprising further steps of:• determining the presence of the glycan first marker on the external surface of particles associated with a second population;• determining the presence of the second marker associated with particles of the second population; and• comparing the results achieved in respect of the first and second markers in the first and second particles, andcharacterising the state of the first and / or second population of particles on the basis of these determinations.

18. A method according to any of claims 4 to 17, further comprising determining the presence of a third (or subsequent) marker in respect of the particles or populations of particles investigated.

19. A method according to any of claims 4 to 18, further comprising determining the presence of markers in third (or subsequent) particles, or third (or subsequent) populations of particles.

20. A method according to any preceding claim, wherein the particles comprise cells, and different first and second cells, or populations of cells, are used to characterise the states of cells from healthy and / or diseased sources.

21. A method according to claim 20, wherein different first and second cells, or populations of cells, are used to characterise the states of cells from sources at different subdivisions in the progression of a disease.

22. A method according to claim 21 , to characterise clinical stages of a disease, such as cancer.

23. A method according to any preceding claim, for use in diagnosis.

24. A method according to any of claims 4 to 19, wherein different first and second cells, or populations of cells, are used to characterise the states of cells from diseased sources when exposed to known or putative treatments for the disease.

25. A method according to claim 24, for use in drug screening.

26. A method according to claim 25, for high throughput screening applications.

27. A method according to any of claims 4 to 19, wherein different first and second cells, or populations of cells, are used to characterise the states of cells from sources at different stages in a disease’s response to treatment.

28. A method according to any of claims 4 to 19, wherein different first and second particles (preferably cells), or populations of particles (preferably cells), are used to characterise the states of cells from different sources having a same disease.

29. A method according to claim 28, wherein the particles (preferably cells), or populations of particles (preferably cells), are derived from different subjects having the same disease.

30. A method according to any preceding claim for use in screening and development of therapeutic agents, or the development of treatment regimens.

31. A method according to any preceding claim, wherein different first and second cells, or populations of cells, may be used to characterise the states of cells from sources at different stages in the progression of a disease.

32. A method according to claim 31 , wherein the cells, or populations of cells, are selected to provide a time course of the progression of the disease.

33. A method according to claim 32, wherein the cells, or population of cells, are selected to provide a time course of the progression of the disease in the absence of treatment.

34. A method according to claim 32 or 33, where information gained regarding the state of such untreated cells, or populations of cells, is compared with information regarding the state of comparable cells undergoing treatment.

35. A method according to claim 34, wherein the comparable cells undergoing treatment may be selected (e.g. by cell sorting) on the basis of their positive response to treatment.

36. A method according to claim any preceding claim, for use to identify how treatment intervenes in the progression of disease to bring about effective therapy.

37. A method according to claim 35, wherein the comparable cells undergoing treatment are selected (e.g. by cell sorting) on the basis of their poor response to treatment.

38. A method according to claim 37, for use to identify how potential treatments fail to provide effective therapy to certain patients.

39. A method according to any preceding claim, wherein the particles comprise cells, and different first and second cells, or populations of cells, are used to characterise the states of cells from healthy and / or diseased sources, and wherein different first and second cells, or populations of cells, are used to characterise the states of cells from sources identified as having genetic alterations associated with a disease.

40. A method according to claim 39, for use to characterise the states of cells from sources having mutations known to be associated with a disease.

41. A method according to claim 40, wherein the mutations are associated with the development of a disease, or with the response of a disease to treatment.

42. A method according to any of any preceding claim, wherein the particles comprise cells, and different first and second cells, or populations of cells, are used to characterise the states of cells from healthy and / or diseased sources, and wherein different first and second cells, or populations of cells, are used to characterise the states of cells from sources identified as having metabolic changes associated with a disease.

43. A method according to claim 42, for use to characterise metabolic changes associated with the development of a disease, or with the response of a disease to treatment.

44. A method according to any preceding claim (preferably according to any of claims 20 to 43) for use in prognosis.

45. A method according to any preceding claim comprising characterising the state of a particle, or of a population of particles, in a number of instances.

46. A method according to claim 45, comprising determining the presence of first and second markers on first and second instances.

47. A method according to claim 46, comprising determining the presence of third (or subsequent) markers on the first and / or second instances, preferably on both the first and second instances.

48. A method according to any of claims 45 to 47, comprising determining the presence of a required number of markers on third (or subsequent) instances.

49. A method according to any preceding claim, comprising characterising the state of a particle, in a method further comprising:• determining the presence of a glycan first marker on the surface of the particle in a first instance and in a second instance;• determining the presence of a second marker associated with the particle in a first instance and in a second instance;• comparing the results achieved in respect of the first and second markers in the first and second instances.

50. A method according to any of claims 2 to 48, comprising characterising the state of a population of particles, in a method further comprising:• determining the presence of a glycan first marker on the surface of the particles within a population in a first instance and in a second instance;• determining the presence of a second marker associated with particles within a population in a first instance and in a second instance;• comparing the results achieved in respect of the first and second markers in the first and second instances.

51. A method according to any of claims 4 to 48, comprising characterising the state of a first and / or second particle, in a method further comprising:• determining the presence of the glycan first marker on the surface of the first particle in a first instance and in a second instance;• determining the presence of the second marker associated with the first particle in a first instance and a second instance;• determining the presence of the glycan first marker on the surface of the second particle in a first instance and in a second instance;• determining the presence of the second marker associated with the second particle in a first instance and a second instance;• comparing the results achieved in respect of the first and second markers, for the first and second particles, in the first and second instances.

52. A method according to any of claims 5 to 48, comprising characterising the state of a first and / or second population of particles, in a method further comprising:• determining the presence of the glycan first marker on the surface of the particles within the first population in a first instance and in a second instance;• determining the presence of the second marker associated with particles within the first population in a first instance and a second instance;• determining the presence of the glycan first marker on the surface of the particles within the second population in a first instance and in a second instance;• determining the presence of the second marker associated with the particles within the second population in a first instance and a second instance;• comparing the results achieved in respect of the first and second markers, for the first and second populations, in the first and second instances.

53. A method according to claim 49 or 51, wherein the determination made in the first instance is made in respect of a first particle, and the determination made in the second instance is made in respect of a second particle.

54. A method according to claim 50 or 52, wherein the determination made in the first instance is made in respect of a first population of particles, and the determination made in the second instance may be made in respect of a second population of particles.

55. A method according to any preceding claim, wherein the glycan markers are determined on the external surface of a particle, or of particles in a population.

56. A method according to any preceding claim, wherein determination in respect of the first and second markers is made on cells.

57. A method according to claim 56, wherein the cells are cells of the same population.

58. A method according to any of claims 56 to 57, wherein the determination in respect of the first and second markers is made in respect of a population of cells that are synchronised with respect to their cell cycles.

59. A method according to any of claims 56 to 58, wherein the determination in respect of the first and second markers is made in respect of a population of cells that are not synchronised with respect to their cell cycles.

60. A method according to any preceding claim, wherein first and second instances are separated by a selected period of time.

61. A method according to claim 60, wherein a stimulus is applied to the particle, or population of particles, during the time between the first and second instances.

62. A method according to claim 60 or 61 , wherein a stimulus is applied to the particle, or population of particles, prior to the first instance.

63. A method according to any preceding claim, wherein a comparison is performed in respect of results achieved at a single time-point.

64. A method according to any of claims 45 to 63, wherein a comparison is performed in respect of results achieved at a plurality of time-points.

65. A method according to claim 64, wherein the comparison is performed in respect of compressed data from the plurality of time-points.

66. A method according to any preceding claim, wherein the comparison comprises analysing a 2D plot of the results; and / or wherein the method comprises providing or generating a 2D plot (optionally a contour plot) of the results, optionally overlaying the 2D plot with a grid; for example where each segment of the grid defines a distinct 2D dimensional space, wherein the state of the particle or population of particles is characterised based on which segment of the grid said particle or population of particles particle (more particularly its / their 2D data point or associated contour) falls within;optionally wherein the method comprises determining the presence of the markers at a first instance and at a second instance, and a 2D plot (or a contour plot) is provided for each of the first and second (and optionally any subsequent) instances; comparing the 2D plot (or contour plot) for each instance, for example wherein the position of a particle (more particularly its 2D data point or associated contour) on a graph for a second instance is compared with the position of said same particle (more particularly its 2D data point or associated contour) on a graph for the first instance; wherein a difference in the position (e.g. dimensional space change) of the particle on the plot for the second instance, relative to its position on the plot for the first instance, indicates a change in particle state between the first and second instances (which may be responsive to an intervention e.g. candidate drug treatment).

67. A method according to claim 66, wherein the comparison comprises analysing a gradient in a 2D plot of the results.

68. A method according to any preceding claim (preferably of any of claims 45 to 67), further comprising characterising the particle or population of particles as:• a particle, or population of particles, in which presence of the first marker and presence of the second marker are correlated with one another; or• a particle, or population of particles, in which presence of the first marker and presence of the second marker are not correlated with one another.

69. A method according to claim 68, wherein the particle or population is characterised as a particle or population in which presence (e.g. level) of the first marker and presence (e.g.level) of the second marker are correlated with one another, and in which the presence of the first marker and presence of the second marker are positively correlated with one another.

70. A method according to claim 68, wherein the particle or population is characterised as a particle or population in which presence (e.g. level) of the first marker and presence (e.g. level) of the second marker are correlated with one another, and in which the presence of the first marker and presence of the second marker are negatively correlated with one another.

71. A method according to claim 69 or claim 70, wherein the first and second marker are correlated with each other in a linear manner.

72. A method according to claim 69 or claim 70, wherein the first and second marker are correlated with each other in a non-linear manner.

73. A method according to claim 68, wherein the particle or population is characterised as a particle or population in which presence (e.g. level) of the first marker and presence (e.g. level) of the second marker are not correlated with one another, wherein the presence of the first marker is variable, and the presence of the second marker is does not vary.

74. A method according to claim 68, wherein the particle or population is characterised as a particle or population in which presence (e.g. level) of the first marker and presence (e.g. level) of the second marker are not correlated with one another, wherein the presence of the first marker does not vary, and the presence of the second marker is variable.

75. A method according to any of claims 45-74 (preferably according to any of claims 60 to 74), wherein an investigative agent of interest is provided to the particles, or populations of particles, prior to the first instance.

76. A method according to any of claims 45-75 (preferably according to any of claims 60 to 75), wherein an investigative agent of interest is provided to the particles, or populations of particles, between the first instance and the second instance.

77. A method according to any of claims 75 to 76, wherein the investigative agent of interest is an inducer of a biological pathway.

78. A method according to claim 77, wherein the investigative agent of interest is an inducer of apoptosis.

79. A method according to claim 78, wherein the inducer of apoptosis is staurosporine.

80. A method according to any of claims 75-77, wherein the investigative agent of interest is a therapeutic agent, or putative therapeutic agent.

81. A method according to any preceding claim wherein the first marker is a glycan marker selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; an N-acetylgalactosamine (GalNAc) residue; a fucose residue; an N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.

82. A method according to claim 81 wherein the glycan first marker is a terminal glycan residue selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; and a terminal N-acetylglucosamine (GIcNAc) residue.

83. A method according to claim 81 or 82, wherein the glycan first marker is terminal sialic acid residue, or a sialic acid residue at the -1 position immediately preceding the terminal glycan residue.

84. A method according to claim any of claims 81-83 (preferably according to claim 83), wherein the sialic acid residue is selected from the group consisting of: an alpha-2, 3-linked sialic acid residue; and an alpha-2, 6-linked sialic acid residue.

85. A method according to claim 81 or 82, wherein the glycan first marker is a terminal galactose residue, or a galactose residue at the -1 position immediately preceding the terminal glycan residue.

86. A method according to claim 81 or 82, wherein the glycan first marker is a terminal mannose residue, or a mannose residue at the -1 position immediately preceding the terminal glycan.

87. A method according to claim 81 or 82, wherein the glycan first marker is a terminal N-acetylgalactosamine (GalNAc) residue, or a GalNAc residue at the -1 position immediately preceding the terminal glycan residue.

88. A method according to claim 81 or 82, wherein the glycan first marker is a terminal fucose residue, or a fucose residue at the -1 position immediately preceding the terminal glycan residue.

89. A method according to claim 81 or 82 wherein the glycan first marker is a terminal N-acetylglucosamine (GIcNAc) residue, or a GIcNAc residue at the -1 position immediately preceding the terminal glycan residue.

90. A method according to any of claims 81 to 89 (preferably according to claim 81), wherein the glycan first marker is an internal N-acetyllactosamine residue.

91. A method according to any preceding claim wherein presence of the glycan first marker is determined by assessing binding of a binding partner for the first glycan marker.

92. A method according to any preceding claim comprising incubating the particle with a binding partner for the first glycan marker.

93. A method according to claim 91 or 92, wherein the binding partner is selected from the group consisting of: a lectin; an antibody, or an antigen-binding fragment thereof; an aptamer; and a reporter cell.

94. A method according to claim 93, wherein the binding partner is a lectin that binds to the glycan first marker (preferably wherein the lectin binds specifically to a single type of first glycan marker).

95. A method according to claim 94, wherein the glycan first marker is a sialic acid residue, and the binding lectin partner is selected from the group consisting of: MAL-II, and SNA.

96. A method according to claim 94, wherein the glycan first marker is a galactose residue, and the binding lectin partner is selected from the group consisting of: PNA, and SJA.

97. A method according to claim 94, wherein the glycan first marker is a mannose residue, and the binding lectin partner is selected from the group consisting of: GNA, and NPL.

98. A method according to claim 94, wherein the glycan first marker is a GalNAc residue, and the binding lectin partner binds to GalNAc.

99. A method according to claim 94, wherein the glycan first marker is a fucose residue, and the binding lectin partner is selected from the group consisting of: AAL, UEA-1 , and LTET.

100. A method according to claim 94, wherein the glycan first marker is a GIcNAc residue, and the binding lectin partner is G.Simp.

101. A method according to claim 94, wherein the glycan first marker is an N-acetyllactosamine residue, and the binding lectin partner is STL.

102. A method according to any of claims 94 to 101, wherein the lectin is a recombinant lectin.

103. Amethod according to any of claims 94-101, wherein the lectin is an animal lectin.

104. Amethod according to claim 103, wherein the lectin is a human lectin.

105. Amethod according to any of claims 94 to 102, wherein the lectin is a plant lectin.

106. A method according to claim 93, wherein the binding partner is an antibody, or an antigen-binding fragment thereof, that binds to the glycan first marker.

107. A method according to claim 106, wherein, the antibody, or antigen-binding fragment thereof, is selected from the group consisting of: a monoclonal antibody; an ScFv antibody fragment; a recombinant Fc fusion; and a camelid antibody.

108. A method according to any of claims 91-107, wherein the binding partner binds to mannose; for example wherein the antibody, or antigen-binding fragment thereof, binds to mannose.

109. A method according to claim 93, wherein the binding partner is a reporter cell.

110. A method according to claim 94, wherein the reporter cell expresses an agent (such as a binding partner used in any of claims 93-108) that binds to a glycan first marker.

111. A method according to any preceding claim wherein the second marker is a glycan marker.

112. A method according to claim 111, wherein the second marker is a glycan marker selected from the group consisting of: a sialic acid residue; a galactose residue; a mannose residue; an N-acetylgalactosamine (GalNAc) residue; a fucose residue; an N-acetylglucosamine (GIcNAc) residue; and an N-acetyllactosamine residue.

113. A method according to claim 112, wherein the second marker is a terminal glycan residue selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; and a terminal N-acetylglucosamine (GIcNAc) residue; or wherein the second marker is selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; a terminal N-acetylglucosamine (GIcNAc) residue; and an internal N-acetyllactosamine residue.

114. A method according to any preceding claim, further comprising determining the presence of a plurality of glycan markers (e.g. two or more glycan markers), and characterising the state of the particle on the basis of these determinations.

115. A method according to claim 114, wherein the presence of three or more glycan markers is determined.

116. A method according to claim 115, wherein the presence of four or more glycan markers is determined.

117. A method according to claim 116, wherein the presence of five or more glycan markers is determined.

118. A method according to claim 117, wherein the presence of six or more glycan markers is determined.

119. A method according to claim 118, wherein the presence of seven or more glycan markers is determined.

120. A method according to claim 119, wherein the presence of eight or more glycan markers is determined.

121. A method according to claim 120, wherein the presence of nine or more glycan markers is determined.

122. Amethod according to claim 121, wherein the presence often or more glycan markers is determined.

123. A method according to any of claims 114 to 122, wherein the plurality of glycan markers are each selected from the group consisting of: a terminal sialic acid residue; a terminal galactose residue; a terminal mannose residue; a terminal N-acetylgalactosamine (GalNAc) residue; a terminal fucose residue; a terminal N-acetylglucosamine (GIcNAc) residue; and an internal N-acetyllactosamine residue.

124. A method according to any preceding claim (preferably according to any of claims 1-110), wherein the second marker is a non-glycan marker.

125. A method according to claim 124, wherein the second marker is selected from the group consisting of: a protein; a lipid; and a nucleic acid.

126. A method according to claim 125, wherein the second marker is a protein.

127. A method according to claim 126, wherein the protein second marker is selected from the group consisting of: a caspase (such as caspase-3); Bcl2; and Bcl2-associated X protein (BAX).

128. A method according to claim 125, wherein the second marker is a lipid.

129. Amethod according to claim 128, wherein the lipid second marker is selected from the group consisting of: phosphatidylserine (PS).

130. A method according to any of claims 124-128, wherein the second marker is selected from the group consisting of: an apoptosis marker; a viability marker; and a cell cycle marker.

131. A method according to claim 130, wherein the apoptosis marker is selected from the group consisting of: a mid-stage apoptosis marker; and an apoptosis regulator protein.

132. Amethod according to claim 131, wherein the mid-stage apoptosis marker is selected from the group consisting of: phosphatidylserine; and a caspase.

133. A method according to claim 129 or 132, wherein presence of phosphatidylserine is determined by binding of a binding partner selected from the group consisting of: annexin V; and an antibody that binds to phosphatidylserine.

134. A method according to claim 131, wherein the apoptosis regulator protein is selected from the group consisting of: Bcl2; and BAX.

135. A method according to claim 130, wherein the viability marker is a nucleic acid.

136. A method according to claim 135, wherein the viability marker is selected from the group consisting of: DNA; and RNA.

137. A method according to claim 135 or 136, wherein the presence of the viability marker is determined by binding of a binding partner selected from the group consisting of: 7-AAD; eF780; SyTox Red; and SyTox Green.

138. A method according to any preceding claim, wherein presence of the first, second, or subsequent, marker is determined by assessing binding of a binding partner for the marker.

139. A method according to claim 138, wherein a binding partner is labelled with a detection moiety.

140. A method according to claim 139, wherein the detection moiety is selected from the group consisting of: a fluorophore; a chromogen; a bead; a tag sequence; biotin; and an enzyme.

141. A method according to claim 140, wherein the flurophore is selected from the group consisting of: phycoerythrin (PE); and propidium iodide (PI).

142. A method according to claim 140, wherein the beads are magnetic beads, and / or beads (such gold, or other metal, beads) able to be detected via microscopy.

143. A method according to claim 140, wherein the enzyme is horseradish peroxidase or luciferase.

144. A method according to any preceding claim, wherein one, more than one, or all of the markers are endogenous markers.

145. A method according to any preceding claim, wherein one, more than one, or all of the markers are exogenous markers.

146. A method according to any preceding claim, wherein the presence of the first, second, or subsequent, marker on the external surface of the particle is determined.

147. A method according to any preceding claim, wherein the presence of the first, second, or subsequent, marker on an excreted particle is determined.

148. A method according to any preceding claim, wherein the presence of each marker is determined with respect to its presence on the external surface of the particle.

149. A method according to any preceding claim, wherein the presence of the second, or subsequent, marker within the particle is determined.

150. A method according to claim 149, wherein the second, or subsequent, marker is the intracellular apoptosis marker mitochondrial membrane potential.

151. A method according to claim 149, wherein the second, or subsequent, marker is an intracellular metabolite.

152. A method according to claim 151, wherein the intracellular metabolite is selected from the group consisting of: free mannose; free pyruvate; ATP; reactive oxygen species; NADH; and NADPH.

153. Amethod according to any one of claims 149-152, wherein the presence of the second marker is determined with reference to an intracellular detection moiety, such as a fluorescent protein.

154. Amethod according to any preceding claim, wherein the determination of the presence of the first marker comprises quantification of the first marker (e.g. thereby providing a level for said first marker).

155. A method according to any preceding claim, wherein the determination of the presence of the second marker comprises quantification of the second marker (e.g. thereby providing a level for said second marker).

156. A method according to any preceding claim, wherein the determination of the presence of both the first and second markers comprises quantification of the first and second markers (e.g. thereby providing a level for said first and second markers).

157. A method according to any preceding claim, wherein the determination of the presence of a third, or subsequent, marker comprises quantification of the marker (e.g. thereby providing a level for said third, or subsequent, marker).

158. A method according to claim 157, wherein determination of the presence of each of the first, second, third, and optionally subsequent markers comprises quantification of the markers (e.g. thereby providing a level for first, second, third, and optionally subsequent markers).

159. A method according to any preceding claim, comprising comparing ratios of the markers.

160. A method according to claim 159, wherein the state of a particle, or population of particles, is characterised by calculating a ratio of the first and second markers.

161. A method according to claim 160, wherein ratios including a third and subsequent markers, if present, is used in characterisation in such an embodiment.

162. A method according to any preceding claim, wherein determination of the presence of the first and second markers is performed by array binding.

163. A method according to any preceding claim (preferably according to any of claims 1-161), wherein determination of the presence of the first and second markers is performed by flow cytometry.

164. A method according to any preceding claim (preferably according to claim 163), wherein determining the presence of a glycan first marker comprises determining the density of the glycan first marker on the external surface of the particle (e.g. based on the surface area of the external surface).

165. A method according to any preceding claim (preferably according to claim 163 or 164), wherein determining the presence of a second marker comprises determining the density of the second marker on the external surface of the particle.

166. A method according to any preceding claim (preferably according to any of claims 163-165), wherein determining the presence of a second marker comprises determining the density of the second marker based on the volume of the particle.

167. A method according to any of claims 163-166, wherein determination of the presence of the first marker is performed by flow cytometry, and wherein determining the presence of a glycan first marker on the external surface of the particle further comprises measuring a level for said glycan first marker on the external surface of the particle; andperforming a moderation step comprising moderating the level for said glycan first marker using a level for a forward scatter signal (FSC level) for the particle; wherein the method optionally comprises actively determining the FSC level as a method step.

168. A method according to claim 167, wherein the moderation step comprises dividing the level for the glycan first marker by the FSC (e.g. FSC-H) level to determine an indication of density of the glycan on the surface (sDen) of the particle.

169. A method according to any of claims 163-168, wherein determination of the presence of the second marker is performed by flow cytometry, and wherein determining the presence of the second marker comprises measuring a level for the second marker on the external surface of the particle; andperforming a moderation step comprising moderating the level for said second marker using a level for a forward scatter signal (FSC level) for the particle.

170. A method according to claim 169, wherein said moderation step comprises dividing the level for the second marker by the FSC level to determine an indication of density of the second marker on the surface of the particle e.g. wherein the second marker is an external marker.

171. A method according to any of claims 163-170, wherein determination of the presence of the second marker is performed by flow cytometry, and wherein determining the presence of the second marker comprises measuring a level for the second marker comprises within the particle’s cell membrane (e.g. for a second marker that is an internal marker); andperforming a moderation step comprising moderating the level for said second marker using a level for a forward scatter signal (FSC) for the particle; wherein the method optionally comprises actively determining the FSC as a method step.

172. A method according to claim 171, wherein said moderation step comprises dividing the level for the second marker by the FSC (e.g. FSC-A) level to the power of 3 / 2 (FSC3 / 2) to determine an indication of volumetric density (vDen) of the second marker within the particle.

173. A method according to any preceding claim wherein the particle comprising a cell membrane is from a prokaryotic source.

174. A method according to claim 173, wherein the particle comprising a cell membrane is from a bacterial source.

175. A method according to any preceding claim wherein the particle comprising a cell membrane is from a eukaryotic source.

176. A method according to claim 175, wherein the particle comprising a cell membrane is from a plant source.

177. A method according to claim 175, wherein the particle comprising a cell membrane is from an animal source.

178. A method according to claim 177, wherein the particle is from a mammalian source.

179. A method according to claim 178, wherein the particle is from a human source.

180. A method according to any of claims 175-178, wherein the particle is from an experimental animal source.

181. A method according to any of claims 177, or 178, or 180 wherein the particle is from a murine source.

182. A method according to any preceding claim, wherein the particle comprising a cell membrane is from a wild-type source.

183. A method according to any of claims 1-181, wherein the particle comprising a cell membrane is from a genetically modified source.

184. A method according to any preceding claim, wherein the particle comprising a cell membrane is selected from the group consisting of: a cell; a cell component (such as a vesicle); and a free cell membrane (such as a ghost).

185. A method according to claim 184, wherein the particle comprising a cell membrane is a cell.

186. A method according to claim 185, wherein the cell is an uncompromised cell.

187. A method according to claim 184 or 185, wherein the cell is a healthy cell.

188. A method according to claim 185, wherein the cell is a pre-apoptotic cell.

189. A method according to claim 185, wherein the cell is an apoptotic cell.

190. A method according to claim 185, wherein the cell is a necrotic cell.

191. A method according to claim 185, wherein the cell is selected from the group consisting of: a blood cell; a stem cell; a neuronal cell; an immune cell; an epithelial cell; an endothelial cell (such as a HUVEC); a bone cell; a muscle cell; a fat cell; a sex cell; a cancer cell; a liver cell; a fibroblast; a cell of an established cell line; and an immortalised cell.

192. A method according to claim 191, wherein the cell is a blood cell selected from the group consisting of: a lymphocyte (such as a T cell or B cell); an erythrocyte; a macrophage; a monocyte; and a granulocyte (such as a neutrophil or eosinophil).

193. A method according to claim 191, wherein the cell is a stem cell selected from the group consisting of: a mesenchymal stem cell; a pluripotent stem cell (such as an iPSC).

194. A method according to claim 185, wherein the cell is a fusion cell or multinucleate cell.

195. A method according to claim 184, wherein the particle comprising a cell membrane is a cell component.

196. A method according to claim 195, wherein the particle comprising a cell membrane is a vesicle.

197. A method according to claim 196, wherein the vesicle is derived from an uncompromised cell.

198. A method according to claim 197, wherein the vesicle is derived from a healthy cell.

199. A method according to claim 196, wherein the vesicle is derived from a pre-apoptotic cell.

200. A method according to claim 196, wherein the vesicle is derived from an apoptotic cell.

201. A method according to claim 196, wherein the vesicle is derived from a necrotic cell.

202. A method according to any of claims 196-201 , wherein the particle is from an animal.

203. A method according to any of claims 195-202, wherein the particle comprising a cell membrane is live.

204. A method according to any of claims 196-203, wherein the particle comprising a cell membrane is not fixed.

205. A method according to any of claims 196-204, wherein the cell membrane of the particle is intact.

206. A method according to any preceding claim, wherein the particle is putatively apoptotic, and the second marker comprises an apoptosis marker.

207. A method according to claim 206, wherein the particle is characterised as being:• Non-apoptotic;• Pre-committed to apoptosis;• Committed to apoptosis; or• Post-committed to apoptosis.

208. A method according to claim 206 or 207, the method comprisingi. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three) glycan markers on the external surface of the particle, ii. comparing each level determined in step (i) with that of a corresponding reference value indicative of non-apoptotic (e.g. healthy) cells to determine a level change for each of the (e.g. two) glycan markers; andHi. characterising the state of the particle on the basis of these comparisons e.g. wherein the state is indicative of a stage of an apoptosis pathway to which the particle belongs.

209. A method according to claim 208, wherein saidat least one (preferably at least two, more preferably at least three) glycan markers are selected from the list consisting of a terminal fucose residue, a terminal mannose residue, and a terminal sialic acid residue; and wherein the particle is characterised as:as a first pre-commitment (to apoptosis) state cell [e.g. about 15 to 30 minutes, or 3%- 10%, through the pre-commitment to apoptosis] when at least one (preferably at least two, more preferably at least three) of the following level changes are determined: o a decrease for internal LacNAc (e.g. a disaccharide having galactose (Gal) and N-acetylglucosamine (GIcNAc) linked by (3-1,4 bonds, or an internal LacNAc bindable by STL), and a decrease for terminal fucose (e.g. bindable byAAL); a second pre-commitment state cell that derives from (or represents a further progression along the biological pathway of) said first pre-commitment state cell (e.g. M1 h pop), when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) for terminal fucose, a decrease (e.g. about 1.9 fold) for terminal mannose, and a decrease (e.g. about 3.1 fold) for terminal sialic acid;a third pre-commitment state cell that derives from (or represents a further progression along the biological pathway of) said second pre-commitment state cell , when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 2.2 fold) for terminal fucose, a decrease (e.g. about 1.4 fold) for terminal mannose, and a decrease (e.g. about 2.0 fold) for terminal sialic acid;a first commitment state cell that derives from (or represents a further progression along the biological pathway of) said third pre-commitment state cell, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 2 fold) for terminal fucose, a decrease (e.g. about 1.1 fold) for terminal mannose, and a decrease (e.g. about 1.6 fold) for terminal sialic acid;a second commitment state cell that derives from (or represents a further progression along the biological pathway of) said first commitment state cell (e.g. M” pop) , when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.4 fold) for terminal fucose, an increase (e.g. about 1.2 fold) for terminal mannose, and substantially no level change for terminal sialic acid;a third commitment (to apoptosis) state particle (e.g. Pop 4) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said second commitment state cell, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 7.4 fold) for terminal fucose, a decrease (e.g. about 4.7 fold) for terminal mannose, and a decrease (e.g. about 5.7 fold) for terminal sialic acid;a fourth commitment state particle (e.g. Pop 5) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said third commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined: o a decrease (e.g. about 5.0 fold) for terminal fucose, a decrease (e.g. about 2.5 fold) for terminal mannose, and a decrease (e.g. about 3.9 fold) for terminal sialic acid;a fifth commitment state particle (e.g. Pop 6) presenting as an apoptotic body state particle that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.1 fold) for terminal fucose, an increase (e.g. about 2.5 fold) for terminal mannose, and an increase (e.g. about 8.4 fold) for terminal sialic acid;a sixth commitment or post-commitment state particle that presents as a necrotic body state particle [e.g. Pop 7] that derives from (or represents a further progression along the biological pathway of) said fifth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.3 fold) for terminal fucose, an increase (e.g. about 2.0 fold) for terminal mannose, and a decrease (e.g. about 51.6 fold) for terminal sialic acid;a seventh commitment or post-commitment state particle presenting as a necrotic body state particle [e.g. Pop 8] that derives from (or represents a further progression along the biological pathway of) said fourth commitment state particle, when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 4.9 fold) for terminal fucose, a decrease (e.g. about 4.1 fold) for terminal mannose, and a decrease (e.g. about 10.8 fold) for terminal sialic acid;an eighth commitment or post-commitment state particle that presents as a vesicle [e.g. Pop 9] that derives from (or represents a further progression along the biological pathway of) said fifth commitment state particle when at least one (preferably at least two, more preferably at least three) of the following level changes are determined: o a decrease (e.g. about 1.5 fold) for terminal fucose, an increase (e.g. about 2.9 fold) for terminal mannose, and an increase (e.g. about 8.3 fold) for terminal sialic acid; ora vesicle state particle [e.g. Pop 10] that derives from blebs of healthy cells (e.g. M pop), when at least one (preferably at least two, more preferably at least three) of the following level changes are determined:o a decrease (e.g. about 1.5 fold) for terminal fucose, an increase (e.g. about 3.1 fold) for terminal mannose, and an increase (e.g. about 9.4 fold) for terminal sialic acid.

210. A method according to claim 208 or 209, whereinthe terminal fucose is bindable by AAL (or wherein the terminal fucose is Fuc (a1,6) GIcNAc);the terminal mannose is bindable by GNA (or wherein the terminal mannose is an alpha-1, 3-linked mannose, such as alpha-1, 3-linked Man3GlcNAc2); andthe terminal sialic acid is bindable by SNA (or wherein the terminal sialic acid is an alpha-2, 6-linked sialic acid, such as NeuAc a2-6 Gal b1-4 GIcNAc).

211. A method according to any one of claims 208-210, wherein the cell is a cell 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).

212. A method according to any one of claims 208-211 , wherein the cell is a stem cell; preferably a mesenchymal stem cell; more preferably a bone marrow mesenchymal stem cell.

213. A method according to any preceding claim, the method comprisingi. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers on the external surface of the particle,ii. comparing each level determined in step (i) with that of a corresponding reference value (preferably density) indicative of CD4-CD8- lymphoid cells (e.g. B cells) to determine a level (preferably density) change for each of the (e.g. at least two) glycan markers; andHi. characterising the state of the particle on the basis of these comparisons e.g. wherein the state is indicative of a stage of a T-cell differentiation pathway to which the particle belongs.

214. A method according to claim 213, wherein said at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers are selected from the list consisting of (i) a terminal N-Acetylglucosamine residue bindable by GSIMP (“GSIMP residue”), a terminal sialic residue bindable by MAL-II (“MAL-II residue”), a terminal sialic residue bindable by SNA (“SNA residue”), a terminal galactose bindable by PNA (“PNA residue”), a terminal fucose bindable by SJA (“SJA residue”), a terminal mannose bindable by NPL (“NPL residue”), a terminal galactose or mannose residue bindable by ConA (“ConA residue”), a terminal fucose bindable by AAL (“AAL residue”), an internal LacNAc residue bindable by STL (“STL residue”); and wherein the particle is characterised as:a CD4-CD8+ T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.4 fold) for the “GSIMP” residue, an increase (e.g.about 2.8 fold) for the “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, an increase (e.g. about 1.1 fold) for the “PNA residue”, a decrease (e.g. about 1.3 fold) for the “SJA residue”, a decrease (e.g. about 1.5 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “NPL residue”, a decrease (e.g. about 1.1 fold) for the “ConA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, and a decrease (e.g. about 1.5 fold) for the “STL residue”;a CD8+(Dim)CD4- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.1 fold) for the “GSIMP” residue, an increase (e.g.about 2.4 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, a decrease (e.g. about 1.3 fold) for the “SJA residue”, an increase (e.g. about 1.3 fold) for the “GNA residue”, an increase (e.g. about 1.4 fold) for the “NPL residue”, an increase (e.g. about 1.1 fold) for the “ConA residue”, an increase (e.g. about 1.3 fold) for the “AAL residue”, and an increase (e.g. about 1.4 fold) for the “STL residue”;a CD8+(High)CD4- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.6 fold) for the “GSIMP” residue, an increase (e.g.about 2.9 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, an increase (e.g. about 1.2 fold) for the “PNA residue”, a decrease (e.g. about 1.3 fold) for the “SJA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.5 fold) for the “NPL residue”, a decrease (e.g. about 1.2 fold) for the “ConA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, and a decrease (e.g. about 1.7 fold) for the “STL residue”;a CD4+CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 3.3 fold) for the “GSIMP” residue, an increase (e.g.about 2.6 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.0 fold) for the “PNA residue”, a decrease (e.g. about 1.2 fold) for the “SJA residue”, a decrease (e.g. about 3.3 fold) for the “GNA residue”, a decrease (e.g. about 3.2 fold) for the “NPL residue”, a decrease (e.g. about 1.4 fold) for the “ConA residue”, a decrease (e.g. about 1.5 fold) for the “AAL residue”, and a decrease (e.g. about 6.5 fold) for the “STL residue”;a CD4+(Dim)CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o an increase (e.g. about 1.3 fold) for the “GSIMP” residue, an increase (e.g. about 1.7 fold) for the “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, an increase (e.g. about 9.7 fold) for the “PNA residue”, a decrease (e.g. about 3.1 fold) for the “SJA residue”, an increase (e.g. about 1.5 fold) for the “GNA residue”, an increase (e.g. about 1.8 fold) for the “NPL residue”, an increase (e.g. about 1.3 fold) for the “ConA residue”, an increase (e.g. about 1.6 fold) for the “AAL residue”, and an increase (e.g. about 4.8 fold) for the “STL residue”; ora CD4+(High)CD8- T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 3.0 fold) for the “GSIMP” residue, an increase (e.g.about 2.6 fold) for the “MAL-II” residue, an increase (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 4.6 fold) for the “PNA residue”, a decrease (e.g. about 1.2 fold) for the “SJA residue”, a decrease (e.g. about 4.1 fold) for the “GNA residue”, a decrease (e.g. about 3.9 fold) for the “NPL residue”, a decrease (e.g. about 1.5 fold) for the “ConA residue”, an decrease (e.g. about 1.5 fold) for the “AAL residue”, and a decrease (e.g. about 7.5 fold) for the “STL residue”.

215. A method according to any preceding claim, the method comprisingi. determining a level (preferably density) for each of at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers on the external surface of the particle,ii. comparing each level determined in step (i) with that of a corresponding reference value (preferably density) indicative of G1 phase cell to determine a level (preferably density) change for each of the (e.g. at least two) glycan markers; andHi. characterising the state of the particle on the basis of these comparisons e.g wherein the state is indicative of the cell cycle stage to which the particle belongs.

216. A method according to claim 215, wherein said at least one (preferably at least two, more preferably at least three, even more preferably at least four) glycan markers are selected from the list consisting of a terminal sialic residue bindable by MAL-II (“MAL-II residue”), a terminal sialic residue bindable by SNA (“SNA residue”), a terminal mannose bindable by GNA (“GNA residue”), a terminal fucose bindable by AAL (“AAL residue”), an internal LacNAc residue bindable by STL (“STL residue”), a terminal galactose bindable by PNA (“PNAresidue”), a terminal galactose or terminal GalNAc bindable by SJA(“SJA residue”), a terminal mannose bindable by NPL (“NPL residue”);wherein the particle is a CD8+(Dim) T cell (e.g. undergoing division) that has been activated by cross-linking CD3 and CD28, and the corresponding reference value (preferably density) is indicative of the first G1 phase of the CD8+(Dim) T cell subsequent to activation;and wherein the particle is characterised as:an S1 phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) forthe “MAL-II” residue, an increase (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 1.2 fold) for the “GNA residue”, an increase (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 2.7 fold) forthe “STL residue”, an increase (e.g. about 1.1 fold) for the “PNA residue”, and an increase (e.g. about 1.3 fold) forthe “SJA residue”, a mid-S phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.9 fold) forthe “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.4 fold) for the “GNA residue”, substantially no level change forthe “AAL residue”, a decrease (e.g. about 3.1 fold) for the “STL residue”, a decrease (e.g. about 1.2 fold) for the “PNA residue”, and substantially no change forthe “SJA residue”; an S2 phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.8 fold) forthe “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.3 fold) forthe “STL residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, and a decrease (e.g. about 1.8 fold) for the “SJA residue”, a G2M S phase (first division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.4 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 1.5 fold) for the “GNA residue”, an increase (e.g. about 1.1 fold) for the “AAL residue”, a decrease(e.g. about 2.7 fold) for the “STL residue”, a decrease (e.g. about 1.5 fold) for the “PNA residue”, and substantially no change for the “SJA residue”;an G1 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) forthe “MAL-II” residue, an increase (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.3 fold) for the “GNA residue”, substantially no level change forthe “AAL residue”, a decrease (e.g. about 2.8 fold) for the “STL residue”, a decrease (e.g. about 1.2 fold) for the “PNA residue”, and an increase (e.g. about 1.5 fold) forthe “SJA residue”, an S1 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2 fold) for the “MAL-II” residue, an increase (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.4 fold) for the “GNA residue”, substantially no level change forthe “AAL residue”, a decrease (e.g. about 3.3 fold) for the “STL residue”, a decrease (e.g. about 1.4 fold) for the “PNA residue”, and a decrease (e.g. about 1.2 fold) forthe “SJA residue”, a mid-S phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.3 fold) forthe “MAL-II” residue, a decrease (about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.8 fold) for the “STL residue”, a decrease (e.g. about 1.7 fold) for the “PNA residue”, and a decrease (e.g. about 1.6 fold) forthe “SJA residue”, an S2 phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.7 fold) forthe “MAL-II” residue, a decrease (about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.3 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.1 fold) forthe “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 2.9 fold) forthe “SJA residue”, a G2M S phase (second division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.3 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.2 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 1.7 fold) for the “SJA residue”; a G1 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.8 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.6 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 3.8 fold) for the “STL residue”, a decrease (e.g. about 1.4 fold) for the “PNA residue”, and a decrease (e.g. about 1.3 fold) for the “SJA residue”, an S1 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.0 fold) for the “MAL-II” residue, substantially no level change for the “SNA residue”, a decrease (e.g. about 1.7 fold) for the “GNA residue”, a decrease (e.g. about 1.1 fold) for the “AAL residue”, a decrease (e.g. about 4.0 fold) for the “STL residue”, a decrease (e.g. about 1.5 fold) for the “PNA residue”, and a decrease (e.g. about 1.5 fold) for the “SJA residue”, a mid-S phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.1 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.4 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 2.3 fold) for the “SJA residue”, an S2 phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.6 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 4.8 fold) for the “SJA residue”,a G2M S phase (third division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.2 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”; an G1 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, substantially no change for the “SNA residue”, a decrease (e.g. about 2.5 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 4.7 fold) for the “STL residue”, a decrease (e.g. about 1.9 fold) for the “PNA residue”, and a decrease (e.g. about 2.6 fold) for the “SJA residue”, an S1 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 2.3 fold) for the “GNA residue”, a decrease (e.g. about 1.2 fold) for the “AAL residue”, a decrease (e.g. about 5.0 fold) for the “STL residue”, a decrease (e.g. about 2.1 fold) for the “PNA residue”, and a decrease (e.g. about 3.2 fold) for the “SJA residue”, a mid-S phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.0 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.7 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.4 fold) for the “STL residue”, a decrease (e.g. about 2.7 fold) for the “PNA residue”, and a decrease (e.g. about 3.8 fold) for the “SJA residue”, an S2 phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.3 fold) for the “SNA residue”, a decrease (e.g. about 2.6 fold) for the “GNAresidue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 4.8 fold) for the “SJA residue”, a G2M S phase (fourth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 2.2 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”; a G1 phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 3.2 fold) for the “GNA residue”, a decrease (e.g. about 1.4 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 2.8 fold) for the “PNA residue”, and a decrease (e.g. about 10.6 fold) for the “SJA residue”; an S1 phase (fifth division post-activation) CD8+(Dim) T cell, when at least two (preferably at least three, more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.7 fold) for the “MAL-II” residue, a decrease (e.g. about 1.1 fold) for the “SNA residue”, a decrease (e.g. about 2.8 fold) for the “GNA residue”, a decrease (e.g. about 1.3 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 3.0 fold) for the “PNA residue”, and a decrease (e.g. about 4.1 fold) for the “SJA residue”; a mid-S phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 1.9 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 3.1 fold) for the “GNA residue”, a decrease (e.g. about 1.5 fold) for the “AAL residue”, a decrease (e.g. about 4.9 fold) for the “STL residue”, a decrease (e.g. about 3.3 fold) for the “PNA residue”, and a decrease (e.g. about 5.9 fold) for the “SJA residue”;an S2 phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.6 fold) for the “MAL-II” residue, a decrease (e.g. about 1.4 fold) for the “SNA residue”, a decrease (e.g. about 3.7 fold) for the “GNA residue”, a decrease (e.g. about 1.6 fold) for the “AAL residue”, a decrease (e.g. about 5.6 fold) for the “STL residue”, a decrease (e.g. about 6.8 fold) for the “PNA residue”, and a decrease (e.g. about 13.7 fold) for the “SJA residue”; ora G2M S phase (fifth division post-activation) CD8+(Dim) T cell, when at least one (preferably at least two, more preferably at least three, even more preferably at least four) of the following level changes are determined:o a decrease (e.g. about 2.2 fold) for the “MAL-II” residue, a decrease (e.g. about 1.2 fold) for the “SNA residue”, a decrease (e.g. about 3.1 fold) for the “GNA residue”, a decrease (e.g. about 1.6 fold) for the “AAL residue”, a decrease (e.g. about 5.2 fold) for the “STL residue”, a decrease (e.g. about 3.5 fold) for the “PNA residue”, and a decrease (e.g. about 3.1 fold) for the “SJA residue”.

217. A method according to any preceding claim, wherein the particle is characterised as being either healthy or diseased.

218. A kit comprising:at least one glycan marker binding partner selected from the group consisting of:i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; and iii) AAL or a binding partner for glycan markers having the same specificity as AAL; and at least one binding partner for a second marker selected from the group consisting of:• a viability marker;• a binding partner for an apoptosis marker.

219. A kit according to claim 218, comprising at least two of the recited glycan binding partners i) to iii).

220. A kit according to claim 219, comprising each of the recited glycan binding partners i) to iii).

221. A kit according to any of claims 218-220, comprising each of the recited binding partners for a second marker.

222. A kit according to any of claims 218-221 , wherein the kit comprises instructions for its use in a method of the invention for detecting cell death.

223. A kit comprising:at least one glycan marker binding partner selected from the group consisting of:i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) NPL or a binding partner for glycan markers having the same specificity as NPL; and iii) MAL II or a binding partner for glycan markers having the same specificity as MAL II; and at least one binding partner for a second marker selected from the group consisting of:• a viability marker;• a binding partner for an apoptosis marker.

224. A kit according to claim 223, comprising at least two of the recited glycan binding partners i) to iii).

225. A kit according to claim 224, comprising each of the recited glycan binding partners i) to iii).

226. A kit according to any of claims 223-225, comprising each of the recited binding partners for a second marker.

227. A kit according to any of claims 223-226, wherein the kit comprises instructions for its use in a method of the invention for detecting cell senescence.

228. A kit comprising:at least one glycan marker binding partner selected from the group consisting of:i) MAL II or a binding partner for glycan markers having the same specificity as MAL II; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; iii) GNA or a binding partner for glycan markers having the same specificity as GNA; iv) AAL or a binding partner for glycan markers having the same specificity as AAL; v) STL or a binding partner for glycan markers having the same specificity as STL; vi) PNA or a binding partner for glycan markers having the same specificity as PNA; and vii) SJA or a binding partner for glycan markers having the same specificity as SJA; and at least one binding partner for a second marker selected from the group consisting of:a viability marker;a binding partner for an apoptosis marker.

229. A kit according to claim 228, comprising at least two of the recited glycan binding partners i) to vii).

230. A kit according to claim 229, comprising at least three of the recited glycan binding partners i) to vii).

231. A kit according to claim 230, comprising at least four of the recited glycan binding partners i) to vii).

232. A kit according to claim 231, comprising at least five of the recited glycan binding partners i) to vii).

233. A kit according to claim 232, comprising at least six of the recited glycan binding partners i) to vii).

234. A kit according to claim 233, comprising each of the recited glycan binding partners i) to vii).

235. A kit according to any of claims 228-234, wherein the kit further comprises at least one further reagent selected from the group consisting of: 4',6-diamidino-2-phenylindole (DAPI); a dye for use in mapping cell division number; and an agent for use in analysing cell proliferation.

236. A kit according to claim 235, wherein the kit comprises at least two of the recited further reagents.

237. A kit according to claim 236, wherein the kit comprises each of the recited further reagents.

238. A kit according to any of claims 235-237, wherein the dye for use in mapping cell division number is CellTrace far red (CTFR).

239. A kit according to any of claims 235-238, wherein the agent for use in analysing cell proliferation is a modified nucleoside (such as 5-ethynyl-2'-deoxyuridine (EDU)).

240. A kit according to any of claims 228-239, wherein the kit comprises instructions for its use in a method of the invention for mapping the cell cycle.

241. A kit comprising:at least one glycan marker binding partner selected from the group consisting of:i) G.Simp or a binding partner for glycan markers having the same specificity as G.Simp; ii) MAL II or a binding partner for glycan markers having the same specificity as MAL II; iii) SNA or a binding partner for glycan markers having the same specificity as SNA; iv) PNA or a binding partner for glycan markers having the same specificity as PNA; v) SJA or a binding partner for glycan markers having the same specificity as SJA; vi) GNA or a binding partner for glycan markers having the same specificity as GNA; vii) NPL or a binding partner for glycan markers having the same specificity as NPL; viii) Con A or a binding partner for glycan markers having the same specificity as Con A; ix) AAL or a binding partner for glycan markers having the same specificity as AAL; and x) STL or a binding partner for glycan markers having the same specificity as STL; and optionally at least one binding partner for a second marker selected from the group consisting of:• a viability marker;• a binding partner for an apoptosis marker.

242. A kit according to claim 241, comprising at least two of the recited glycan binding partners i) to x); or at least two of the recited glycan binding partners i)-vii) and ix)-x).

243. A kit according to claim 242, comprising at least three of the recited glycan binding partners i) to x); or at least three of the recited glycan binding partners i)-vii) and ix)-x).

244. A kit according to claim 243, comprising at least four of the recited glycan binding partners i) to x); or at least four of the recited glycan binding partners i)-vii) and ix)-x).

245. A kit according to claim 244, comprising at least five of the recited glycan binding partners i) to x); or at least five of the recited glycan binding partners i)-vii) and ix)-x).

246. A kit according to claim 245, comprising at least six of the recited glycan binding partners i) to x); or at least six of the recited glycan binding partners i)-vii) and ix)-x).

247. A kit according to claim 246, comprising at least seven of the recited glycan binding partners i) to x); or at least seven of the recited glycan binding partners i)-vii) and ix)-x).

248. A kit according to claim 247, comprising at least eight of the recited glycan binding partners i) to x); or at least eight of the recited glycan binding partners i)-vii) and ix)-x).

249. A kit according to claim 248, comprising at least nine of the recited glycan binding partners i) to x); or at least nine of the recited glycan binding partners i)-vii) and ix)-x).

250. A kit according to claim 249, comprising each of the recited glycan binding partners i) to x); or each of the recited glycan binding partners i)-vii) and ix)-x).

251. Akit according to any of claims 241-250, comprising one of the recited binding partners for a second marker.

252. A kit according to claim 251, comprising each of the recited binding partners for a second marker.

253. A kit according to any of claims 241-252, wherein the kit comprises instructions for its use in a method of any of claims 1-217 for detecting cell differentiation.

254. Akit comprising:at least one glycan marker binding partner selected from the group consisting of:i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) NPL or a binding partner for glycan markers having the same specificity as NPL; iii) Con A or a binding partner for glycan markers having the same specificity as Con A; iv) SNA or a binding partner for glycan markers having the same specificity as SNA; v) MAL II or a binding partner for glycan markers having the same specificity as MAL II; vi) AAL or a binding partner for glycan markers having the same specificity as AAL; vii) UEA-1 or a binding partner for glycan markers having the same specificity as UEA-1 ; viii) LTET or a binding partner for glycan markers having the same specificity as LTET; ix) PNA or a binding partner for glycan markers having the same specificity as PNA; x) SJA or a binding partner for glycan markers having the same specificity as SJA; xi) STL or a binding partner for glycan markers having the same specificity as STL; and xii) G.Simp or a binding partner for glycan markers having the same specificity as G.Simp; and optionally at least one binding partner for a second marker selected from the group consisting of:• a viability marker;• a binding partner for an apoptosis marker.

255. A kit according to claim 254, comprising at least two of the recited glycan binding partners i) to xii); or at least two of the recited glycan binding partners i)-ii) and iv)-xii).

256. A kit according to claim 255, comprising at least three of the recited glycan binding partners i) to xii); or at least three of the recited glycan binding partners i)-ii) and iv)-xii).

257. A kit according to claim 256, comprising at least four of the recited glycan binding partners i) to xii); or at least four of the recited glycan binding partners i)-ii) and iv)-xii).

258. A kit according to claim 257, comprising at least five of the recited glycan binding partners i) to xii); or at least five of the recited glycan binding partners i)-ii) and iv)-xii).

259. A kit according to claim 258, comprising at least six of the recited glycan binding partners i) to xii); or at least six of the recited glycan binding partners i)-ii) and iv)-xii).

260. A kit according to claim 259, comprising at least seven of the recited glycan binding partners i) to xii); or at least seven of the recited glycan binding partners i)-ii) and iv)-xii).

261. A kit according to claim 260, comprising at least eight of the recited glycan binding partners i) to xii); or at least eight of the recited glycan binding partners i)-ii) and iv)-xii).

262. A kit according to claim 261, comprising at least nine of the recited glycan binding partners i) to xii); or at least nine of the recited glycan binding partners i)-ii) and iv)-xii).

263. A kit according to claim 262, comprising at least ten of the recited glycan binding partners i) to xii); or at least ten of the recited glycan binding partners i)-ii) and iv)-xii).

264. A kit according to claim 263, comprising at least eleven of the recited glycan binding partners i) to xii); or at least eleven of the recited glycan binding partners i)-ii) and iv)-xii).

265. A kit according to claim 264, comprising each of the recited glycan binding partners i) to xii); or each of the recited glycan binding partners i)-ii) and iv)-xii).

266. A kit according to any of claims 254-265, comprising one of the recited binding partners for a second marker.

267. A kit according to claim 266, comprising each of the recited binding partners for a second marker.

268. A kit according to any of claims 254-267, wherein the kit comprises instructions for its use in a method of the invention.

269. A kit comprising:at least one glycan marker binding partner selected from the group consisting of:i) GNA or a binding partner for glycan markers having the same specificity as GNA; ii) SNA or a binding partner for glycan markers having the same specificity as SNA; iii) AAL or a binding partner for glycan markers having the same specificity as AAL; iv) PNA or a binding partner for glycan markers having the same specificity as PNA; and v) STL or a binding partner for glycan markers having the same specificity as STL; and optionally at least one binding partner for a second marker selected from the group consisting of:• a viability marker;• a binding partner for an apoptosis marker.

270. A kit according to claim 269, comprising at least two of the recited glycan binding partners i) to v).

271. A kit according to claim 270, comprising at least three of the recited glycan binding partners i) to v).

272. A kit according to claim 271, comprising at least four of the recited glycan binding partners i) to v).

273. A kit according to claim 272, comprising each of the recited glycan binding partners i) to vii).

274. A kit according to any of claims 269-273, comprising one of the recited binding partners for a second marker.

275. A kit according to claim 274, comprising each of the recited binding partners for a second marker.

276. A kit according to any of claims 269-275, wherein the kit comprises instructions for its use in a method of the invention for detecting cell death.

277. A kit according to any of claims 218-276, wherein the binding partner for a nucleic acid marker is 7AAD.

278. A kit according to any of claims 218-277, wherein the binding partner for an apoptosis marker is a binding partner for phosphatidylserine.

279. A kit according to claim 278, wherein the binding partner for phosphatidylserine is Annexin V.

280. A kit according to any of claims 218-279, wherein one or more of the binding partners is labelled with a detection moiety.

281. A kit according to claim 280, wherein the detection moiety is a fluorophore, or a conjugation moiety.

282. A kit according to claim 281 , wherein the conjugation moiety is biotin.

283. A kit according to any of claims 218-282, further comprising a detection moiety coupled to a conjugation moiety.

284. A kit according to claim 283, wherein the detection moiety is a fluorophore.

285. A kit according to claim 284, wherein the fluorophore is phycoerythrin.

286. A kit according to any of claims 283-285, wherein the conjugation moiety is streptavidin.

287. A kit comprising a binding partner (e.g. lectin) for binding a first glycan biomarker selected from the group consisting ofi) a terminal sialic acid residue,ii) a terminal galactose residue,iii) a terminal mannose residue,iv) a terminal N-acetylgalactosamine (GalNAc) residue,v) a terminal fucose residue,vi) a terminal N-acetylglucosamine (GIcNAc) residue, andvii) an internal N-acetyllactosamine (LacNAc) residue;and a binding partner for binding a second non-glycan marker, wherein the second marker is selected from the group consisting of: an apoptosis marker; a viability marker; a cell division marker; a cell differentiation marker; a cell cycle marker; a disease marker (whether an acquired disease or an inherited disease); and a cell biology marker.

288. A kit according to any of claims 218-287, wherein at least of the constituents is provided in lyophilised form.

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