Methods for moderating flow cytometry data, and computer program product

Abstract

Methods for moderating flow cytometry data for cells and / or other particles based on the size of said cells and / or other particles are provided, for example for cell-state determination. In particular, the method discloses 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, and a forward scatter signal, FSC, for each of the one or more particles, and 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 using the forward scatter signal. Preferably, the indication of the marker present comprises an indication of a sugar or glycan molecule present in the one of more particles.

Description

[0001] Methods for moderating flow cytometry data

[0002] Field of the invention

[0003] The present disclosure relates to computer-implemented methods for moderating flow cytometry data for cells and / or other particles based on size of said cells and / or other particles, in particular based on surface area or volume of said cells and / or other particles.

[0004] Background

[0005] 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.

[0006] 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 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.

[0007] 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.

[0008] 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.

[0009] The flow cytometer data also typically undergoes gating. Many gates, which are methodsof 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.

[0010] The compensated data, in a gated subpopulation, may then be used for analysis, typically presented in either 1 D (histograms) or 2D plots.

[0011] Summary of the invention

[0012] Aspects of the invention are as set out in the independent claims and optional features are set out in the dependent claims. Aspects of the invention may be provided in conjunction with each other and features of one aspect may be applied to other aspects.

[0013] An aspect of the invention relates to a computer-implemented method for moderating flow cytometry data. The method comprises 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, and a forward scatter signal, FSC, for each of the one or more particles. The method then comprises moderating the indication of the marker present for one or more cells, vesicles, and / or specific cell bodies within the sample based on an indication of size for said one or more particles using the forward scatter signal.

[0014] The one or more particles may be biological particles, including but not limited to cells, vesicles, specific cell bodies, organelles, and / or other biological particles comprising a membrane. Alternatively, or in addition, the particles may be non-biological particles such as beads, for example including beads that have been bound or dosed with one or more markers configured to be detected with a flow cytometer.

[0015] 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 byfactors 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.

[0016] 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.

[0017] 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 marker within the one or more particles (for example wherein the indication of the marker present comprises an indication of an intracellular marker).

[0018] 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.

[0019] 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.

[0020] 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, 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, cellsurface 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.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 the sample 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, or a ubiquitous class or set of molecules, 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. For example, a plurality of markers may be used to provide an indication of a ubiquitous class or set of molecules. 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.

[0021] 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.

[0022] 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 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, etc.

[0023] For example, a selection of non-limiting examples have been included below.

[0024] (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 V present by the forward scatter signal to determine an indication of surfacedensity of 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.

[0025] (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.

[0026] (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 density of lectin for the one or more particles (e.g. cells, vesicles, and / or specific cell bodies etc).

[0027] 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.

[0028] 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.

[0029] For example, moderating the indication of the marker present for one or more particleswithin 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)3 / 2, to determine an indication of volumetric density (vDEN) of the marker within the one or more particles.

[0030] 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).

[0031] 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.

[0032] 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.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.

[0033] 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.

[0034] 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:

[0035] 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;

[0036] obtaining an indication of size for the one or more particles; and

[0037] moderating the indication of the marker present for one or more particles within the sample by the indication of size.

[0038] 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 onimage analysis, for example including Al-based techniques.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 against surface area. This method may, in some examples, unify analysis of Lectome density across all cells and vesicles with varying cell size.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.

[0044] 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 V marker.

[0045] 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 / markers may be used.

[0046] 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.

[0047] 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.

[0048] 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 flowcytometer 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. 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.

[0049] 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.

[0050] Another aspect of the invention provides for a computer-implemented method of identifying dead cell subpopulations within a biological sample. The method comprises obtaining flow cytometer data for a population of particles (such as cells, vesicles, and / or specific cell bodies) within a biological sample, wherein the population have been marked with a viability marker, such that the flow cytometer data comprises an indication of the viability marker present for each particle within the population. The method also comprises obtaining an indication of size for each particle, and moderating the indication of the viability marker present for each particle using the indication of size for said particle. Finally, the method comprises identifying a first subpopulation of particles, based on the corrected indication of the viability marker, wherein the first subpopulation of particles are considered to be dead.

[0051] 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.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.

[0052] The viability marker may comprise, but is not limited to, at least one of:

[0053] (i) Annexin V, and wherein identifying the first subpopulation of particles based on the corrected indication of the Annexin V marker comprises identifying the first subpopulation of particles to be dead by apoptosis; or

[0054] (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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Another aspect of the invention provides for a computer program product comprising instructions configured to program a programmable processor to perform the method of any preceding aspects of the invention.Another aspect of the invention provides for a graphical representation of a plurality of marker for a cell or vesicle comprising:

[0059] 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

[0060] 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.

[0061] For example, the plurality of markers may comprise one or more surface lectins or other terminal sugar recognising molecule / marker.

[0062] Each circular sector may be assigned a colour, wherein the colour represents a type of marker or lectin.

[0063] 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).

[0064] Drawings

[0065] Embodiments of the disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0066] Fig. 1 shows a schematic to illustrate the effect of moderation of flow cytometer data based on size.

[0067] Fig. 2 shows a box diagram of an example system of the invention.

[0068] Fig. 3 shows a flow diagram of an example method of the present invention.

[0069] Fig. 4A shows a plot of flow cytometry data without moderation by size for a specific sample. Fig. 4B shows a plot of the same flow cytometry data of Fig. 4A, wherein the data on the y-axis has been moderated according to the method of Fig. 3, based on the FSC signal. Fig. 4C shows a plot of the same flow cytometry data of Fig. 4A, wherein the data on the x-axis has been moderated according to the method of Fig. 3, based on the FSCsignal. Fig. 4D shows a plot of the same flow cytometry data of Fig. 4A, wherein the data on the x-axis and the y-axis has been moderated according to the method of Fig. 3, based on the FSC signal.

[0070] Fig. 5A shows a plot of flow cytometry data without moderation by size for another specific sample; whilst Fig. 5B shows a plot of the same flow cytometry data of Fig. 5A, wherein the data on both axes has been moderated according to the method of Fig. 3.

[0071] Fig. 6 shows two sets of comparative plots comparing flow cytometry data based on lectin surface markers in both “raw”, unmoderated form, and moderated form according to the method of Fig. 3. Each set of comparative plots relates to a respective cell type, subjected to the same treatments at various time points.

[0072] Fig. 7 shows a subset of the data from Fig. 6 being subjected to confirmatory viability analysis.

[0073] Fig. 8 shows a flow diagram of another example method of the present invention.

[0074] Fig. 9 shows a flow diagram of another example method of the present invention, for example wherein the method of Fig. 9 is an example of a method according to Fig. 8.

[0075]

[0076] Embodiments of the claims relate to computer-implemented methods and systems for moderating flow cytometry data based on size.

[0077] It will be appreciated from the discussion above that the embodiments shown in the Figures are merely exemplary, and include features which may be generalised, removed, or replaced as described herein and as set out in the claims.

[0078] 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.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.

[0079] The problem arising from existing flow cytometry techniques which do not moderate based on size is illustrated in a schematic Fig. 1.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] FIG. 2 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Fig. 3 shows a flow diagram of an example method of the invention, for example for use with the system of Fig. 2.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Moderation may be applied on an individual cell / particle basis, or on a population basis.This is also discussed in more detail below.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Furthermore, the processor 204 is preferably configured to identifying at least one population or subpopulation of cells or other biological bodies / particles within the sample based on the moderated indication of the markers present.

[0096] Surface density normalisation, sDEN

[0097] This moderation equation addresses the surface marker’s relationship to the surface area of a cell.

[0098] 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.

[0099] FSC ∝ cross sectional area = πr2

[0100] The cells and / or other particles are approximated to have a surface area of an assumed sphere (A = 4πr2), meaning surface area is directly proportional to cross-sectional area. It therefore follows that FSC is directly proportional to surface area.

[0101] This provides:

[0102] Signal

[0103] sDEN surface density) = ks

[0104] FSC

[0105] wherein ksis a constant and can be ignored when comparing between cell or other particles.

[0106] For a population or subpopulation:population average fluorescence signal

[0107] sDEN = - ■ — ■_ - - - population average surface area

[0108] For example:

[0109] population average fluorescence

[0110] sDEN = -; — ■_ - — — - - —

[0111] population average FSC — A

[0112] 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.

[0113] For example, if marker A is measured with the fluorochrome, FITC, population average FITC — A

[0114] sDEN = - - — ■_ - — — - - population average FSC — A

[0115] where “average” could be mean fluorescence intensity or geometric fluorescence intensity.

[0116] At a single cell level,

[0117] single cell fluorescence

[0118] sDEN = — sin —gli -e cellT s —urf 7 -ace area

[0119] As such, if marker A is measured with the fluorochrome, FITC,

[0120] single cell FITC — A

[0121] sDEN = — —i - / 7TTT - T

[0122]

[0123] single cell FSC — A

[0124] 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.

[0125] Whilst the above equation illustrates one method to address a surface marker’s relationship to the surface area of a cell, the skilled person will understand that other methods or equations may be used, such as, for example, equations based upon Mie or Fraunhofer diffraction.

[0126]

[0127] Volumetric \ vDEN

[0128] When considering intracellular markers, the calculation of density may need to be adjustedto account for cellular volume rather than cross-sectional or surface area. Since,

[0129] 4

[0130] Volume =4 / 3πr3

[0131] 3

[0132] 3 3

[0133] Volume ∝ (Area)3 / 2∝ (FSC)3 / 2

[0134] This provides:

[0135] Signal

[0136] vDEN (volumetric density) = kvSignal / (FSC)3 / 2

[0137] wherein kvis a constant and can be ignored when comparing between cell or other particles.

[0138] For the population,

[0139] population average of fluorescence

[0140] vDEN = population average of fluorescence / (population average cross sectional area)3 / 2

[0141] population average of fluorescence

[0142] = population average of fluorescence / (population average FSC)3 / 2

[0143] For the single cell,

[0144] single cell fluorescence

[0145] vDEN = single cell fluorescence / (single cell surface area)3 / 2

[0146] single cell fluorescence

[0147] = single cell fluorescence / (single cell FSC)3 / 2

[0148] While the equations themselves may appear trivial, the size normalisation provides a type of fundamental normalisation I moderation of flow cytometry analysis, that has been omitted since the invention of flow cytometry.

[0149] 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 isapplied.

[0150] Merely by way of example:

[0151] • 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:

[0152] Raw 7AAD

[0153] DNA content Density [7AAD] = Raw 7AAD / (FSC - A)3 / 2

[0154] • Lectin markers: If cell-surface lectin markers are used, moderating such may comprise:

[0155] Raw Lectomic signal

[0156] Lectomic Density = - — — - - -

[0157]

[0158] Alternatively, the skilled person will understand that if intracellular lectin markers are used, moderating such may comprise:

[0159] Raw Lectomic signal

[0160] Lectomic Density = Raw Lectomic signal / (FSC - A)3 / 2

[0161] • Annexin V If Annexin V markers are used, moderating such may comprise:

[0162] Raw AnnexinV

[0163] AnnexinV Density = Raw AnnexinV / FSC - A

[0164] 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.

[0165] For example,

[0166] A Lectin binding

[0167] Specific Lectin Density = - — — -

[0168]

[0169] and / orΔ AnnexinV binding

[0170] Specific AnnexinV Density = - — — -

[0171]

[0172] where Δ Lectin or Δ Annexin-V binding are calculated by subtracting Lectin or Annexin-V control binding from respective raw binding signals. However, the skilled person will understand that this is not limited to only lectin or Annexin-V markers, and that the same may be calculated for other markers.

[0173] 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.

[0174] Example 1 - Necrosis and Apoptosis analysis

[0175] Fig. 4A 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 V 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.

[0176] 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.

[0177] As shown in Fig. 4A, 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.

[0178] Fig. 4B 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 V is shown on the x-axis. In contrast to Fig. 4A, a clear-cut discriminationthreshold 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.

[0179] Fig. 4C 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 V is shown on the x-axis. Fig. 4D 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 density-moderated data relating to marker Annexin V is shown on the x-axis. When Annexin V data is moderated according to density, improvements in clarity of subpopulations separated by Annexin V density boundaries is observed compared to Annexin V raw boundaries. By way of illustration, a first sub-population boundary is indicated by arrow 412, and a second sub-population boundary is indicated by arrow 414. In both instances, the boundary becomes more defined when Annexin V 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] Example 2 - Cell vesicle analysis

[0184] Since vesicles are approximately one tenth the size of a normal cell, the impact of densitycorrection becomes great. This sensitivity to density is illustrated by Figs. 5A and 5B which compare traditional raw fluorescence-based axis plotting (Fig. 5A) versus new densitybased axis plotting (Fig. 5B). 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.

[0185] In the example shown in Figs. 5A and 5B, cells are treated for 4 hours with a drug which is expected to induce much vesicle formation during apoptosis.

[0186] Fig. 5A shows an example plot of flow cytometer data wherein “raw” data relating to marker Annexin V 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.

[0187] Fig. 5B shows an example plot of the same flow cytometer data as Fig. 5A, wherein surface density moderated (sDen) data relating to marker Annexin V 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.

[0188] Comparing the raw plot of Fig. 5A with the density-moderated plot of Fig. 5B, 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.

[0189] Example 3 - Apoptotic body analysis

[0190] Another example which highlights the impact of density moderation of the present invention is shown in Fig. 6. In particular, the density moderated data reveals the appearance of a new subpopulation that was obscured completely by raw fluorescencebased analysis methods.

[0191] 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 V is shown on the x-axis, and “raw” data relating to various lectin surface markers (LSM) is shown on the y-axis. The plotsillustrate marker expression for vehicle control 610, Cell Type 1 cells after an 8-hour drug treatment time (Staurosporine, ST) 612, and Cell Type 1 cells after a 16-hour drug treatment time (Staurosporine, ST) 614.

[0192] 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 surface density (sDen) of marker Annexin V 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 (Staurosporine, ST) 612, and 16-hour drug treatment time (Staurosporine, ST) 614.

[0193] 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 V 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 (ST) 618, and the Cell Type 2 cells after a 16-hour drug treatment time (ST) 620.

[0194] The plots shown in column 608 plot the same flow cytometer data for the same population of Cell Type 2 cells wherein density-moderated data relating to the surface density (sDen) of marker Annexin V 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 (ST) 618, and 16-hour drug treatment time (ST) 620.

[0195] A new 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 created by 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 markercan completely change when density is applied.

[0196] Further to Fig. 6, Fig. 7 illustrates data obtained from subsequent testing that illustrates that the new population 704 is not necrotic by using normalised data obtained using a viability dye. Line 702 indicates the start of the new population in all density plots. Plot 700S provides an example plot using sDEN for the viability dye, and plot 700V provides a comparative example plot using vDEN viability dye normalisation. In both plots 700S and 700V, viable populations are identified to the right hand side of line 702.

[0197] 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.

[0198] Fig. 8 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.

[0199] 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.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.

[0200] 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 volume can be approximated, for example such as radius, diameter, circumference, cross sectional area, etc.

[0201] As discussed above, two primary types of moderation are provided: (i) surface density normalisation (sDEN), and (ii) volumetric density normalisation (vDEN).

[0202] Moderation may also be applied on an individual cell I particle basis, or on a population basis, as discussed above.

[0203] By way of example, Fig. 9 illustrates an alternative method wherein an approximation of the area of a cell or other particle (including but not limited to cells, vesicles, specific cell bodies, or other biological particles) may be determined based on images of said cell or body.

[0204] In this example, the processor 204 obtains flow cytometer data from the flow cytometer 202, wherein the flow cytometer data comprises an indication of a marker present for a set of cells and / or other particles within a sample, and a set of images corresponding to the set of cells and / or other particles within a sample.

[0205] The processor 204 then obtains an indication of size for each of the set of cells and / or other particles within a sample based on the set of images. For example, the area of a cell / body may be determined from flow cytometry and image cytometry based on counting the pixels in a cell / body image.

[0206] If image area, Ai, is defined as the area observable from an image of the cell or otherbiological body / particle obtained from some form of cytometry or microscopy (such as the number of pixels present in the cell or particle), then a proportional interpretation of the estimated cell surface area, As, can be assigned as:

[0207] As∝ AI

[0208] The processor then moderates the indication of the marker present within the sample based on the indication of size (930).

[0209] Following the above, moderating the indication of the marker based on surface density can be determined as:

[0210] fluorescence or other indication of biomarker for cell or particle SDEN = - - - - -; —;; - - - - - — -

[0211]

[0212] number of pixels in image of cell or particle

[0213] Estimated volume of the cell, assuming perfect sphere, V, can be approximated as:

[0214] 3

[0215] V ∝ A3 / 2

[0216] As such, moderating the indication of the marker based on volumetric density can then be determined as:

[0217] fluorescence or other indication of biomarker for cell or particle vDEN = - j -

[0218]

[0219] (number of pixels in image of cell or particle^

[0220] The invention disclosed herein can be applied to sugar signal moderation, such as moderation of terminal sugar signals, for the purpose of, for example, cell-state determination. The disclosed moderation process, applied to ubiquitous molecules, such as sugar molecules, may provide some advantages over selecting a specific marker. For example, moderating from two or more sugars, or other ubiquitous molecules, on the same cell surface can confirm the same cell / particle from two or more signals. Ubiquitous molecules may describe a class of surface-located, dynamically varying, universally-expressed, species and cell type agnostic, ubiquitous molecules. This may be advantageous in comparison to methods utilising single markers. For example, specific protein markers can only be used for cells that have said specific marker on the surface and therefore cannot normalise between cells in a sample which do not have the specific protein. The use of sugar signal moderation provides a ubiquitous method of differentiatingbetween any and / or all cells in a population. Such a method is not possible using protein recognition on cell surfaces. The method disclosed herein may also provide a moderation method suitable for use with live cells.

[0221] Lectin-based markers may be advantageous for providing an indication of a sugar molecule, such as a terminal sugar molecule, present. 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, etc.

[0222] The method may also advantageously be applied for flow cytometry data including two or more markers, wherein each marker is configured to provide an indication of a different molecule present. Preferably, at least one of the two or more markers may be configured to provide an indication of a sugar present, or other ubiquitous molecule. Preferably, the method may be performed using a plurality of markers configured to provide indications of different sugars and / or glycans present. More preferably, a first one or more markers may be configured to provide an indication of sugar and / or glycan molecules, and a second, or subsequent, marker(s) may be configured to provide an indication of a different molecule present, for example such as an indication of a protein, lipid, DNA, or other molecule, etc. This may be advantageous as one or more markers for indications of sugars, including terminal or sub-terminal sugars, and / or glycans may be particularly useful for cell state determination, for example where, whereas the “other” marker(s) can be used to identify variability in expression of a molecule associated with said “other” marker across cell states.

[0223] For example, in some examples, the first marker may provide an indication of a terminal mannose. This may be advantageous as this marker has been seen to increase in senescence of a variety of cell types. However, the skilled person will understand that this is merely one example and is not intended to be limiting.

[0224] By way of example only, the Attune CytPix from Thermofisher is one example flow cytometer that allows brightfield images to be obtained along with other traditional flow parameters. Alternatively, imagestream™ (CyTek) offers both brightfield and fluorescence images of cells. However, the skilled person will understand that these examples are notintended to be limiting.

[0225] The description above often refers to analysis of cells, however the skilled person will understand that the methods disclosed herein also apply to analysis derived from flow cytometer data for other non-cell particles, such as vesicles (blebs), specific cell bodies, and / or other biological bodies or particles. This is intended to also include apoptotic bodies and emerging apoptotic-necrotic transit populations. Indeed, the methods may be particularly advantageous for analysis of such non-cell biological matter due to differences in size which aims to be accounted and compensated for in the present methods. Furthermore, the skilled person will understand that the methods disclosed herein also apply to flow cytometer data for non-biological particles, including but not limited to flow cytometer beads and the like.

[0226] In the context of the present disclosure other examples and variations of the apparatus and methods described herein will be apparent to a person of skill in the art.

[0227] Embodiments of the disclosure are set out in the following numbered clauses:

[0228] 1. A computer-implemented method for moderating flow cytometry data, the method comprising:

[0229] 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, and a forward scatter signal, FSC, for each of the one or more particles;

[0230] 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 using the forward scatter signal.

[0231] 2. The method of any preceding clause wherein moderating the indication of the marker present for one or more particles within the sample using the forward scatter signal comprises 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.

[0232] 3. The method of any preceding clause wherein moderating the flow cytometry datacomprises 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.

[0233] 4. The method of clause 3 wherein the indication of the marker present for one or more particles comprises an indication of a surface marker present for the one or more particles.

[0234] 5. The method of any preceding clause wherein moderating the flow cytometry data comprises 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.

[0235] 6. The method of clause 5 wherein the indication of the marker present for one or more particles comprises an indication of an intracellular marker present for the one or more particles.

[0236] 7. The method of any preceding clause comprising 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.

[0237] 8. The method of any of clauses 1 to 6 comprising moderating the flow cytometry data for a plurality of particles by:

[0238] determining an average indication of the marker present for said plurality of particles;

[0239] 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.

[0240] 9. The method of clause 8 wherein the plurality of particles is a population of cells, vesicles, and / or specific cell bodies within the sample, for example including a subpopulation of cells, vesicles, and / or specific cell bodies within the sample, optionally wherein the method further comprises gating the flow cytometer data to identify the population of cells, vesicles, and / or specific cell bodies within the sample.10. The method of any preceding clause wherein the indication of the marker present for one or more particles within the sample comprises an indication of fluorescence of the one or more particles within the sample, wherein said marker is configured to fluoresce.

[0241] 11. The method of any preceding clause wherein the indication of the marker present comprises 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.

[0242] 12. The method of any preceding clause wherein the indication of the marker present for one or more particles within the sample comprises at least one of:

[0243] (i) an indication of Annexin V, wherein moderating the flow cytometry data comprises dividing the indication of Annexin V present by the forward scatter signal to determine an indication of surface density of Annexin V for the one or more particles;

[0244] (ii) 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; and / or

[0245] (iii) an indication of a terminal sugar recognising molecule, such as a lectin, wherein moderating the flow cytometry data comprises dividing the indication of the terminal sugar recognising molecule present by the forward scatter signal to determine an indication of surface density of said terminal sugar recognising molecule for the one or more particles.

[0246] 13. The method of any preceding clause wherein the indication of the marker present for one or more particles within the sample comprises an indication of a number of the marker present for the one or more particles within the sample.

[0247] 14. The method of any preceding clause further comprising:

[0248] obtaining flow cytometer data for a control sample, 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

[0249] determining size calibration data which associates FSC signals with particle size, based on the obtained flow cytometer data for the control sample;

[0250] wherein moderating the indication of the marker present for one or more particles withinthe sample based on an indication of size for said one or more particles uses the forward scatter signal and the size calibration data.

[0251] 15. The method of clause 14 wherein moderating the indication of the marker present for one or more particles within the sample further comprises:

[0252] 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; optionally wherein dividing the indication of the marker present based on the indication of size comprises:

[0253] (i) dividing the indication of the marker present by the indication of size to determine an indication of surface density of the marker for the one or more particles; and / or

[0254] (ii) dividing the indication of the marker present by the indication of size to the power of 3 / 2, (the indication of size)3 / 2, to determine an indication of volumetric density of the marker within the one or more particles.

[0255] 16. A computer-implemented method for graphical analysis of flow cytometry data analysis, the method comprising:

[0256] performing the method for moderating flow cytometry data of any of clauses 1 to 15; and

[0257] graphically plotting the moderated indication of the marker present for one or more particles within the sample.

[0258] 17. The method of clause 16 wherein graphically plotting the moderated indication of the marker present comprises 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.

[0259] 18. The method of clause 17 wherein plotting the moderated indication of the second marker present for the same one or more particles comprises plotting lectin density datafor the same one or more particles within the sample on the second axis; optionally wherein the indication of the marker present for the one or more particles comprises an indication of lipids present, such that graphically plotting the moderated indication of the marker present comprises plotting lectin density data for the one or more particles within the sample on the second axis, and plotting the moderated lipid density data for the same one or more particles within the sample on the first axis.

[0260] 19. A computer-implemented method for moderating flow cytometry data for a sample, the method comprising:

[0261] 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;

[0262] obtaining an indication of size for the one or more particles within the sample; and moderating the indication of the marker present for one or more particles within the sample by the indication of size.

[0263] 20. A computer-implemented method of identifying cell subpopulations within a biological sample, the method comprising:

[0264] obtaining flow cytometer data for a population of particles within a biological sample, wherein the flow cytometer data comprises:

[0265] (i) a first indication of a first marker present for each particle within the population of particles;

[0266] (ii) a second indication of a second marker present for each particle within the population of particles, wherein the second indication is obtained based on at least one lectin marker; and

[0267] 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;

[0268] moderating the second indication of the second marker present for each particle, using indication of size for said particle;

[0269] 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; and

[0270] identifying a first subpopulation of particles within the population of particles based on the graphical plot.21. A computer-implemented method of identifying dead cell subpopulations within a sample, the method comprising:

[0271] obtaining flow cytometer data for a population of particles within a sample, wherein the population of particles have been marked with a viability marker, such that the flow cytometer data comprises an indication of the viability marker present for each particle within the population of particles;

[0272] obtaining an indication of size for each of the particles;

[0273] moderating the indication of the viability marker present for each particle using the indication of size for said particle;

[0274] identifying a first subpopulation of particles based on the corrected indication of the viability marker, wherein the first subpopulation of particles are considered to be dead.

[0275] 22. The method of clause 21 wherein the viability marker comprises at least one of:

[0276] (i) Annexin V, and wherein identifying the first subpopulation of particles based on the corrected indication of the Annexin V marker comprises identifying the first subpopulation of particles to be dead by apoptosis; or

[0277] (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.

[0278] 23. The method of any of clauses 19 to 22 wherein 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.

[0279] 24. The method of any of clauses 19 to 23 wherein the indication of size is obtained using image analysis of at least one image of the one or more particles within the sample.

[0280] 25. The method of clause 24 wherein the flow cytometer data for the sample comprises imaging flow cytometer data comprising at least one image of the one or more particles within the sample, wherein obtaining the indication of size for the one or more particles within the sample comprises determining cell size based on image analysis of the at least one image.26. A computer program product comprising instructions configured to program a programmable processor to perform the method of any preceding clause.

Claims

CLAIMS:

1. A computer-implemented method for moderating flow cytometry data, the method comprising: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, and a forward scatter signal, FSC, for each of the one or more particles;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 using the forward scatter signal;wherein the indication of the marker present comprises an indication of a sugar or glycan molecule present in the one of more particles.

2. The method of any preceding claim wherein moderating the indication of the marker present for one or more particles within the sample using the forward scatter signal comprises 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.

3. The method of any preceding claim wherein moderating the flow cytometry data comprises 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.

4. The method of claim 3 wherein the indication of the marker present for one or more particles comprises an indication of a surface marker present for the one or more particles.

5. The method of any preceding claim wherein moderating the flow cytometry data comprises 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.

6. The method of claim 5 wherein the indication of the marker present for one or more particles comprises an indication of an intracellular marker present for the one or more particles.

7. The method of any preceding claim comprising 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.

8. The method of any of claims 1 to 6 comprising 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.

9. The method of claim 8 wherein the plurality of particles is a population of cells, vesicles, and / or specific cell bodies within the sample, for example including a subpopulation of cells, vesicles, and / or specific cell bodies within the sample, optionally wherein the method further comprises gating the flow cytometer data to identify the population of cells, vesicles, and / or specific cell bodies within the sample.

10. The method of any preceding claim wherein the indication of the marker present for one or more particles within the sample comprises an indication of fluorescence of the one or more particles within the sample, wherein said marker is configured to fluoresce.

11. The method of any preceding claim wherein the flow cytometer data comprises an indication of a second marker present for the one or more particles within the sample; and moderating the indication of the second marker present for one or more particles within the sample based on an indication of size for said one or more particles using the forward scatter signal, for example wherein the indication of the second marker present comprises 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, wherein the second marker is different to the first marker for a sugar or glycan molecule.

12. The method of any preceding claim wherein the indication of the marker present for one or more particles within the sample comprises at least one of:(i) an indication of Annexin V, wherein moderating the flow cytometry data comprises dividing the indication of Annexin V present by the forward scatter signal to determine an indication of surface density of Annexin V for the one or more particles;(ii) 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; and / or(iii) an indication of a terminal sugar recognising molecule, such as a lectin, wherein moderating the flow cytometry data comprises dividing the indication of the terminal sugar recognising molecule present by the forward scatter signal to determine an indication of surface density of said terminal sugar recognising molecule for the one or more particles.

13. The method of any preceding claim wherein the indication of the marker present for one or more particles within the sample comprises an indication of a number of the marker present for the one or more particles within the sample.

14. The method of any preceding claim further comprising:obtaining flow cytometer data for a control sample, 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; anddetermining size calibration data which associates FSC signals with particle size, based on the obtained flow cytometer data for the control sample;wherein 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 uses the forward scatter signal and the size calibration data.

15. The method of claim 14 wherein moderating the indication of the marker present for one or more particles within the sample further comprises: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; optionally wherein dividing the indication of the marker present based on the indication ofsize comprises:(i) dividing the indication of the marker present by the indication of size to determine an indication of surface density 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)3 / 2, to determine an indication of volumetric density of the marker within the one or more particles.

16. A computer-implemented method for graphical analysis of flow cytometry data analysis, the method comprising:performing the method for moderating flow cytometry data of any of claims 1 to 15; andgraphically plotting the moderated indication of the marker present for one or more particles within the sample.

17. The method of claim 16 wherein graphically plotting the moderated indication of the marker present comprises 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.

18. The method of claim 17 wherein plotting the moderated indication of the second marker present for the same one or more particles comprises plotting lectin density data for the same one or more particles within the sample on the second axis; optionally wherein the indication of the marker present for the one or more particles comprises an indication of lipids present, such that graphically plotting the moderated indication of the marker present comprises plotting lectin density data for the one or more particles within the sample on the second axis, and plotting the moderated lipid density data for the same one or more particles within the sample on the first axis.

19. A computer-implemented method for moderating flow cytometry data for a sample, the method comprising:obtaining flow cytometer data for a sample, the flow cytometer data comprising anindication of a marker present for one or more particles within the sample;obtaining an indication of size for the one or more particles within the sample; and moderating the indication of the marker present for one or more particles within the sample by the indication of size.

20. A computer-implemented method of identifying cell subpopulations within a biological sample, the method comprising:obtaining flow cytometer data for a population of particles within a biological sample, wherein the flow cytometer data comprises:(i) a first indication of a first marker present for each particle within the population of particles;(ii) a second indication of a second marker present for each particle within the population of particles, wherein the second indication is obtained based on at least one lectin marker; andobtaining 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 indication of the second marker present for each particle, using indication of size for said particle;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; andidentifying a first subpopulation of particles within the population of particles based on the graphical plot.

21. A computer-implemented method of identifying cell subpopulations within a biological sample, the method comprising:obtaining flow cytometer data for a population of particles within a biological sample, wherein the flow cytometer data comprises:(i) a first indication of a first marker present for each particle within the population of particles;(ii) a second indication of a second marker present for each particle within the population of particles, wherein the second marker is configured to provide an indication of a sugar present for each particle within the population ofparticles; andobtaining 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 indication of the second marker present for each particle, using indication of size for said particle;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; andidentifying a first subpopulation of particles within the population of particles based on the graphical plot.

22. A computer-implemented method of identifying dead cell subpopulations within a sample, the method comprising:obtaining flow cytometer data for a population of particles within a sample, wherein the population of particles have been marked with a viability marker, such that the flow cytometer data comprises an indication of the viability marker present for each particle within the population of particles;obtaining an indication of size for each of the particles;moderating the indication of the viability marker present for each particle using the indication of size for said particle;identifying a first subpopulation of particles based on the corrected indication of the viability marker, wherein the first subpopulation of particles are considered to be dead.

23. The method of claims 22 wherein the viability marker comprises at least one of:(i) Annexin V, and wherein identifying the first subpopulation of particles based on the corrected indication of the Annexin V marker comprises identifying the first subpopulation of particles to be dead by apoptosis; or(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.

24. The method of any of claims 19 to 23 wherein the flow cytometer data further comprises a forward scatter signal, FSC, for each particle, wherein the indication of sizeis obtained based on the forward scatter signal for each particle.

25. The method of any of claims 19 to 24 wherein the indication of size is obtained using image analysis of at least one image of the one or more particles within the sample.

26. The method of claim 25 wherein the flow cytometer data for the sample comprises imaging flow cytometer data comprising at least one image of the one or more particles within the sample, wherein obtaining the indication of size for the one or more particles within the sample comprises determining cell size based on image analysis of the at least one image.

27. A computer program product comprising instructions configured to program a programmable processor to perform the method of any preceding claim.

Images