WO2008081198A1

WO2008081198A1 - Blood assays for predicting inflammatory responses

Info

Publication number: WO2008081198A1
Application number: PCT/GB2008/000033
Authority: WO
Inventors: Trevor T. Hansel
Original assignee: Imperial Innovations Ltd
Priority date: 2007-01-05
Filing date: 2008-01-07
Publication date: 2008-07-10

Abstract

To provide in vitro assays for modelling the human side effects of drugs, two procedures are disclosed. Firstly, a rapid fixing protocol is provided that can improve the ability to assay cell surface markers of cell activation, allowing them to be quantified while still present at high levels. This fixing protocol is particularly useful when working with whole blood, thereby permitting immune cell responses to be assessed in the presence of endogenous plasma proteins and the like. Secondly, the CD11b cell surface marker can be used as a predictive indicator of undesired inflammatory activity. Substances can thus be tested in vitro against human blood cells, particularly substances that are not known or expected to provoke an inflammatory response, with up regulated expression of CD11b indicating a potential in vivo problem. These two approaches can be used separately or in combination.

Description

BLOOD ASSAYS FOR PREDICTING INFLAMMATORY RESPONSES

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of inflammatory assays.

BACKGROUND ART

In March 2006, the first human clinical trials of TGN1412, a monoclonal antibody with anti-CD28 super-agonist activity, resulted in a cytokine release syndrome (cytokine storm) and severe inflammatory response in the six subjects. A capillary leak syndrome led to multiple organ failure, necessitating treatment on the intensive care unit. All subjects are now discharged from hospital, but one has severe ischaemic damage of the fingers and toes. The effects were unexpected because the drug had been tested at 50Ox the tested human dose in the usual animal tests for toxicology and safety, and they demonstrate the need for in vitro assays for modelling the human side effects of drags. The final report from an expert group investigating the TGNl 412 trial reported that an in vitro assay based on measuring cytokine release after incubating liquid phase TGN1412 with whole blood or with PBMCs for 48 or 72 hours did not predict any problems.

It is an object of the invention to address the need for further and improved in vitro safety assays. It is a further object of the invention to provide in vitro safety assays that can predict cytokine release before it happens, rather than measure cytokine release after it has already occurred.

DISCLOSURE OF THE INVENTION The invention meets this object in a two different ways. Firstly, a rapid fixing protocol is provided that can improve the ability to assay cell surface markers of cell activation, allowing them to be quantified while still present at high levels. This fixing protocol is particularly useful when working with whole blood, thereby permitting immune cell responses to be assessed in the presence of endogenous plasma proteins and a range of blood cells as well as added drugs. Secondly, the CDl Ib cell surface marker can be used as an indicator of undesired inflammatory activity. CDl Ib is present in resting leukocytes (white blood cells) largely as a preformed protein in granules within the cytoplasm. On leukocyte activation this preformed cytoplasmic granule CDl Ib rapidly mobilises onto the leukocyte cell surface. An assay of CDl Ib can thus be used to predict that a downstream cytokine storm may occur. Substances can thus be tested in vitro against human blood cells, particularly substances that are not known or expected to provoke an inflammatory response, with up-regulated expression of CDlIb predicting a potential in vivo problem. These two approaches can be used separately or in combination.

Thus a first aspect of the invention provides a method for fixing and labelling a cell surface molecule of a cell, comprising steps of: (i) applying a stimulus to the cell; then (ii) contacting the cell with a surface molecule fixative; and then (iii) contacting the cell with a labelling reagent. Unlike existing labelling protocols where surface markers are labelled and then fixed, this method fixes the markers and then labels them. It has been found that this order of steps (a) allows the method to be performed on cells in the presence of whole blood, rather than merely on isolated leukocytes or mononuclear cells, and (b) can permit higher signal levels to be seen, particularly for markers that have been found to be down-regulated during labelling e.g. CDl Ib. Similarly, the invention provides a method for fixing surface molecules of a cell, comprising steps of: (i) applying a stimulus to the cell; and then (ii) contacting the cell with a surface molecule fixative, wherein the method does not include a step of labelling cell surface markers between steps (i) and (ii).

A second aspect of the invention provides an in vitro method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of: (i) contacting the substance with a cell; and (ii) assessing the effect of step (i) on cell surface CDlIb expression by the cell. An increase in CDl Ib expression in response to stimulation by the substance indicates cellular activation and a possible downstream inflammatory response.

Combining both of these aspects of the invention, the invention provides an in vitro method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of:

(i) contacting the substance with a cell; (ii) contacting the cell with a fixative; and (iii) contacting the cell with a labelling reagent for CDlIb. The results of labelling in step (iii) can be used to assess the effect of step (i) on CDl Ib expression by the cell. A pro-inflammatory compound can cause a rapid increase in CDl Ib, and this increase can readily be detected. The cell will usually be a blood cell, and the fixative will usually be a CDl Ib fixative.

Similarly, the invention provides an in vitro method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of: (i) contacting the substance with a cell; (ii) contacting the cell with a fixative; and (iii) contacting the cell with a labelling reagent for CDl Ib, wherein the method does not include a step of labelling cell surface markers between steps (i) and (ii). The invention also provides a method comprising the steps of a method as disclosed above, for use in assessing the possible in vivo anti-inflammatory effects of a substance. It also provides the use of a method of the invention for assessing the possible in vivo anti-inflammatory effects of a substance.

As a variation of the second aspect, the invention can be used to see in vitro if a substance may have desired or undesired in vivo anti-inflammatory effects. In this variation, a cell (typically a blood cell) is provided with a potential anti-inflammatory substance prior to a 'positive control' inflammatory stimulus, to see if the inflammatory response is inhibited. Thus the invention provides a method for assessing the possible in vivo anti-inflammatory effects of a substance, comprising the steps of: (i) contacting a cell with an potential anti-inflammatory substance then (ii) contacting the cell with an inflammatory stimulus and then (iii) assessing the effect of step (i) and (ii) on CDl Ib expression by the cell. A decrease in CDl Ib expression in response to stimulation by the substance indicates inhibition of inflammation. The invention also provides a method for assessing the possible in vivo anti-inflammatory effects of a substance, comprising the steps of: (i) contacting a cell with an potential anti-inflammatory substance then (ii) contacting the cell with an inflammatory stimulus and then (iii) contacting the cell with a CDl Ib fixative; and (iv) contacting the cell with a labelling reagent for CDl Ib. The methods of the invention can rapidly reveal leukocyte activation. Thus they can be used to identify potentially harmful in vivo side effects, and so may help to avoid repeating the unfortunate events seen with TGNl 412. Furthermore, by providing an assay that can assess effects in whole human blood, closely matching in vivo situations, the invention permits a reduction in animal testing prior to human clinical trials. Indeed, the assays can be performed at an early stage during research, screening, development and/or optimisation of human pharmaceuticals and may avoid effort that will ultimately prove to have been wasted.

Stimulating cells

Where a method of the invention involves a step of applying a stimulus to the cell, various stimuli can be used, including physical stimuli (e.g. electromagnetic radiation, sound waves, etc.) and/or chemical stimuli. The use of such stimuli in methods for investigating the behaviour of cells is well known. The term "applying a stimulus to the cell" does not mean that the cell must necessarily be stimulated as a result; rather, it means that the cell is exposed to a potential stimulus, with actual stimulation occurring only if the cell is responsive to the stimulus. Thus the invention includes methods in which a cell fails to respond to an applied stimulus. Typically the method will involve the use of one or more chemical stimuli. Suitable substances that may be used for stimulating the cells include, but are not limited to: antibodies; lipopolysaccharides; cytokines; chemokines; small organic molecules; polypeptides; polysaccharides; immunomodulators; recombinantly-expressed proteins; allergens; nucleic acids; vaccines; pharmaceutical excipients or additives; preservatives; imaging agents; etc. Antibodies and small organic molecules are preferred substances for use with the invention.

Where the invention investigates the effect of an antibody, any form of antibody can be used. Thus the invention can use polyclonal antibodies or monoclonal antibodies. It can use human antibodies, non-human antibodies, chimeric antibodies, CDR-grafted antibodies, humanised antibodies, etc. It can use full-length native antibodies or fragments thereof, such as Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, etc. It can use single-chain Fv (scFv) antibodies, including multiple linked scFvs known as diabodies or triabodies. It can use single variable domain or VHH antibodies, such as those found in camelids and sharks. It can use domain antibodies (dAbs). It can use antibodies with a K or λ light chain. It can use anti-idiotypic antibodies. It can use antibodies with a α, δ, ε, γ or μ heavy chain. It can also use antibodies known as "superantibodies", which typically comprise an antibody (as defined above) covalently linked to at least one non-antibody moiety which has catalytic, adjuvant, membrane-penetrating, and/or autophilic properties.

A "small organic molecule" has a molecular weight <2000 Da e.g. <1500 Da, <1000 Da, <750 Da, <500 Da, etc., but typically more than 100 Da. Small organic molecules will typically include at least one aromatic group, such as an aromatic hydrocarbon moiety, an aromatic heterocycle, etc. Small organic molecules may include, for instance, one or more of a benzene, naphthalene, anthracene, phenanthrene, pyrrole, fiiran, thiophene, imidazole, pyrazole, oxazole, thiazole, pyridine, indole, benzofuran, thionaphthene, quinoline, and/or isoquinoline group. Such molecules may be derived from large libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from various suppliers, or may be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures.

This technique can also be used to predict potential inflammatory effects of nucleic acids, including DNA and RNA and vectors for use in genetic engineering, gene therapy, etc. Substances applied as stimuli to cells will usually not be expected, before the methods of the invention are performed, to provoke an inflammatory response. Rather, the methods are used to identify unexpected inflammatory effects of substances. Thus a substance may have been tested in animal models (e.g. including primate models) without having revealed a pro-inflammatory activity, and the methods of the invention are used to see if the same absence of pro-inflammatory activity can be expected in humans.

Similarly, the effect of a substance on the expression of CDlIb will usually not be known before the methods of the invention are performed.

In some embodiments of the invention, the substance is not a CXCLl (also known as GRO-α). hi some embodiments of the invention, the substance is not a CXCL5 (also known as ENA-78). hi some embodiments of the invention, the substance is not a CXCL8 (also known as interleukin-8). In some embodiments of the invention, the substance is not a CCL2 (also known as GDCF-2 and MCPl). Thus, in some embodiments, the substance may not be an interleukin, or may not be an intercrine, or may not be a cytokine, or may not be a chemokine.

In some embodiments of the invention, the substance is not SB-656933 e.g. the substance may not be a selective CXCR2 antagonist, or may not be a CXCR2 antagonist.

Cells can be exposed to stimuli for short periods e.g. up to 6 hours e.g. up to about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, etc. Treatments shorter even than 20 minutes can be used. Short treatments, particularly for 1 hour or less, minimise artefacts caused by cell isolation procedures, and with minimal intrinsic platelet and monocyte activation. Cells will usually be exposed to stimuli at about body temperature e.g. between 34 and 40⁰C, between 35 and 39°C, between 36 and 38°C, or at 37°C. Surface molecule fixatives

A method of the invention may involve a step of contacting a cell with a surface molecule fixative. Various fixatives can be used, including fixatives used in existing protocols.

Preferred fixatives include cross-linking reagents, such as formaldehyde, glutaraldehyde, glyoxal, p-phenylenediglyoxal, etc. These will usually be used as aqueous solutions e.g. formaldehyde may be used in the form of formalin, and maybe prepared using paraformaldehyde.

A fixative that has been found to be particularly useful is a dilute aqueous solution of formaldehyde. Such solutions may include from 0.05% to 1% (by volume) of formaldehyde e.g. between 0.1% and 0.5% formaldehyde, between 0.2% and 0.3% formaldehyde, or about 0.25% formaldehyde. An aqueous medium used to dilute a cross-linker will preferably be buffered e.g. with a phosphate buffer, such as phosphate-buffered saline.

Where a fixative is used after a step of cell stimulation, it is preferably used very soon after. For instance, the fixative may be contacted with the cells within 1 hour of the end of cell stimulation e.g. within 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, or sooner. When analysing a molecule that can rapidly be re-internalised from the cell surface {e.g. CDl Ib) then fixation within 5 minutes of the end of stimulation {e.g. <5, <4, <3, <2 or even <1 minute after the end) is preferred, in order to maximise signal intensity and accuracy.

The duration of fixation can be similarly short e.g. 30 minutes, 20 minutes, 10 minutes, 5 minutes, or shorter. A 1 minute fixing step has been found to be adequate for analysing CDlIb after 10 minutes of stimulation of whole blood.

Fixatives are preferably used at low temperatures, below room temperature. Thus they may be used at between -10 and 1O⁰C, at between -5 and 5°C, at between 0 and 4°C, etc. Low temperatures help to preserve cell surface structure during labelling.

Fixatives will generally not be specific to a particular surface molecule, but will fix a variety of different surface molecules. Preferred cell surface fixatives are able to fix at least surface-expressed CDl Ib, in order to substantially prevent loss of CDlIb from the cell surface e.g. via shedding or re-internalisation by the cell. Treatments with ice-cold dilute formaldehyde (0.25%) solution have been found to fix CDl Ib suitably.

Fixation of surface CDl Ib has previously been reported (Hamblin et al. (1992) J Immunol Methods 146:219-28) by immediately treating cells with buffered formaldehyde, but this prior art method prevents the cells from up-regulating surface expression of CDl Ib in response to stimuli. Advantageously, the methods of the invention fix CDl Ib after stimulation has taken place. Surface molecules

A method of the invention may involve the labelling of a cell surface molecule. It may also involve fixing of the cell surface molecule prior to its labelling. The fixing and labelling permit subsequent analysis (including quantitative analysis) of the amount of the molecule e.g. by FACS analysis. Rather than analysing all cell surface molecules, methods will usually be concerned with specific target molecule(s). The surface molecule(s) of interest will depend on the type of cell and the effect being analysed. For instance, if T cell activation is being analysed then the CD25 protein may be used as a cell surface marker i.e. the method may fix and label CD25. CDl Ib can be used to analyse activation of leukocytes, including neutrophils, eosinophils, monocytes, T cells, B cells, NK cells and other leukocyte populations .

CDlIb is a component of β₂-integrins. It has also been named CR-3 alpha chain, αMβ₂, pi 70, cell surface glycoprotein MAC-I alpha subunit, ITGAM (integrin alpha-M), leukocyte adhesion receptor MOl, neutrophil adherence receptor, and integrin alpha M (complement component 3 receptor 3 subunit). It is a single-pass type I membrane protein and the reference sequence for the human protein is found in GenBank under reference GI:88501734.

CDl Ib is found both pre-formed in cytoplasmic granules and on the cell surface of leukocytes. Upon cellular activation, the cytoplasmic stores are mobilised and are rapidly displayed on the cell surface, where it acts as part of a leukocyte cell adhesion molecule and also as a complement receptor. The quick display means that surface levels of CDl Ib are very variable and fluctuate rapidly. After being exposed on the surface, CDl Ib can be re-internalised by cells, and it has now been found that CDl Ib levels decrease during labelling of cells by standard methods. Thus the amount of labelled CDl Ib present after labelling is not an accurate reflection of the actual amount of CDlIb that was present at the start of labelling, and the signal-to-noise ratio is also reduced. To avoid these problems with CDl Ib labelling, the invention provides a procedure in which CDl Ib is first fixed on the cell surface and is then labelled.

To assess the effect of a substance on surface expression of CDl Ib by a cell, the level of surface expression is measured after exposure to the substance and is compared to the level before exposure to the substance. The comparison will qualitatively reveal if CDlIb levels have increased, decreased or stayed the same. The comparison may additionally provide quantitative information about the amount of any increase/decrease. Thus the methods of the invention may rely on relative and/or absolute measurements of CDl Ib levels. The skilled person can readily select an appropriate baseline for the comparison. For instance, the baseline may be the expression level on the actual treated cell before being exposed to the substance, or it may be a parallel control cell which is not exposed to the substance. The baseline may have been determined before performing a method of the invention, or may be determined during (e.g. in parallel) or after the method has been performed. As an alternative it may be an absolute baseline derived from previous work. A preferred method for quantitatively analysing levels of cell surface CDl Ib expression involves flow cytometry (e.g. FACS). FACS assays for quantitative analysis of CDl Ib have been disclosed by Furebring et al. (2006) Scandinavian Journal of Immunology 63:208-216, by Casilli et al. (2005) Biochemical Pharmacology 69:385-394, by Bnmialti et al. (2006) Shock 25:351-357, and by Nicholson et al. (2007) Pulmonary Pharmacology & Therapeutics 20: 52-59. The method of Nicholson et al. (2007) is preferred.

Cells

Methods of the invention are performed on cells, and in particular on blood cells. Although single cell assays are possible (e.g. see WO2006/117541), the methods of the invention will typically be performed on a population of cells e.g. at least 10⁵ cells. Moreover, they will usually be performed on a population of mixed cell types, and the invention advantageously provides methods that can be performed on whole blood. Thus signals from neutrophils, for instance, can be detected and analysed despite the presence of other blood cells. By looking at whole blood, rather than merely at isolated leukocytes or mononuclear cells, immune cell responses can be assessed in the presence of endogenous plasma proteins and the like, giving a closer model of in vivo situations. This situation contrasts with the usual procedure of isolating leukocyte and mononuclear cells prior to their assessment (e.g. Furebring et al. (2006), Casilli et al. (2005), etc.).

Whole human blood will typically be freshly-obtained human blood. The blood should not have been treated or manipulated in any way, except that anti-coagulants may be added (e.g. EDTA, heparin, etc.). Starting with fresh blood, stimuli may be applied for up to 2 hours at 37⁰C, thus minimising artefacts caused by cell isolation procedures, and with minimal platelet and monocyte activation. Blood should ideally be taken slowly and gently (e.g. not with a vacutainer system), and subjected to as little agitation and pipetting as possible, in order to minimise any intrinsic activation. CDl Ib levels on unstimulated leukocytes are very low, but even small amounts of mis-handling and damage of blood cells causes levels to increase rapidly (i.e. will increase the baseline levels of CDl Ib and reduce the signal-to-noise ratio after deliberate stimulation).

Analysis of CDl Ib expression on neutrophils in whole human blood is preferred.

Inflammatory stimuli

Some methods of the invention involve the use of a positive stimulus of an inflammatory response. Many such stimuli are known in the art, including various cytokines and chemokines, and any of these can be used. For instance, the cell may be exposed to a suitable amount of tissue necrosis factor (TNF-α), LPS (endotoxin), etc.

Labelling reagents

A method of the invention may involve a step of contacting a cell with a labelling reagent. Various labelling reagents can be used, including those used in existing protocols. The labelling reagent will lead to modification of a molecule in a cell such that it can be detected more easily. For detection purposes, a labelling reagent will include a detectable moiety. This moiety will give a detectable signal, either intrinsically or when suitable stimulated, permitting its detection. Suitable detectable moieties include dyes (including both chromophores and fluorophores, such as fluorescein or phycoerythrin, etc.), luminescent groups, radioactive isotopes, enzymes (as in ELISA, to convert substrates into detectable products), etc. It is also possible to include a functional group that will itself permit the downstream attachment of a label e.g. a biotin group, an avidin group, a streptavidin group, etc.

Dyes and stains themselves are usually non-selective as labels i.e. they label many different target molecules, such as all cell surface proteins rather than just the ones of interest. For methods of the invention, however, a labelling reagent will usually be selective i.e. it will label substantially only target molecules of interest. Thus a labelling reagent will include a targeting moiety. Typical targeting moieties are antibodies, including monoclonal antibodies (usually from a non-human animal, such as a rat, mouse, goat or rabbit). Preparation of labelled antibodies specific for surface markers of interest is a routine technique in this field. Preferring labelling reagents for use with molecules on the surface of whole cells are fluorescently- labelled monoclonal antibodies. For labelling and detection of CDlIb expression, for instance, a monoclonal anti-CDl lb carrying a fluorescent FITC (fluorescein isothiocyanate) label can be used. Labelled anti-CDl lb antibodies are commercially available from both Invitrogen™ and Abeam™. For instance, anti-CDl Ib antibodies are available from Invitrogen labelled with FITC (catalogue ref. AHSl 128), biotin (AHSl 129), phycoerythrin (AHSl 147) pacific blue (RM2828), etc., and 37 different anti-CDl Ib antibodies are available from Abeam.

Where a cell of interest is present in a population of mixed cells {e.g. a neutrophil in a sample of whole blood) and where a target molecule of interest is present on multiple cell types (e.g. CDlIb, which is present inter αliα on neutrophils and monocytes), then a second labelling reagent may be used in order to allow signals from different cell types to be distinguished. For instance, when using a CDl Ib label it is possible to include a CD 16 label (to identify neutrophils) or a CD 14 label (to identify monocytes). The second labelling reagent will give a different signal from the first (e.g. it will cany a different fluorophore) and this second signal can be used to divide the signal from the CDlIb label according to cell type. A second labelling reagent may also be used to distinguish nucleated cells (e.g. leukocytes) from un-nucleated cells (e.g. erythrocytes), such as LDS-751 dye.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessaiy, the word "substantially" may be omitted from the definition of the invention. The term "about" in relation to a numerical value x means, for example, x+10%.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

BRIEF DECSRIPTION OF DRAWINGS

Figures 1 and 2 show CDlIb surface expression levels measured by FACS, showing % expression relative to baseline levels. Figure 1 shows levels in neutrophils activated with chemokines, and figure 2 shows levels in monocytes. The symbols represent different stimulating chemokines: CXCL8 (■), CXCL5 (T), CXCLl (A) and CCL2 (♦). Data are means ± SE.

Figure 3 shows similar results, but using a CXCR2 antagonist. Symbols show data from healthy patients (■) or patients with COPD (A).

Figure 4 shows the CDl Ib signal from FACS analysis of neutrophils stimulated with LPS.

MODES FOR CARRYING OUT THE INVENTION CDlIb FACS assay to assess leukocyte activation

Blood was collected from patients by venepuncture, and blood handling was performed with the minimum of disturbance. Blood was anticoagulated with sterile 0.25M EDTA and used immediately.

Cells were activated with inflammatory cytokines CXCLl, CXCL5, CXCL8 and CCL2. Whole blood (100 ml) was incubated at 37°C for 10 min with either lOμl chemokine (at Hx final whole blood concentration) or buffer. The samples were placed on ice and ice-cold 0.25% formaldehyde solution (250 ml; "Cellfϊx") was added to fix cell surface molecules. After 1 minute, 40 ml was removed and added to a 1.8 ml Eppendorf tube containing FITC-labelled anti-CDl lb monoclonal antibody (10 ml). For specifically measuring CDlIb upregulation in neutrophils, the tube also contains PE-labelled anti-CD16 (5 ml); for specifically measuring CDl Ib upregulation in monocytes, the tube also contains PE-labelled anti-CD 14 (5 ml). A control sample-containing FITC- labelled mouse anti-IgG2a (10 ml) or the anti-CD16 or anti-CD14 antibodies (5 ml) was also prepared. The tubes were gently mixed and left on ice in the dark for 20 min. After staining with antibody the blood was added to cold DPBS (0.5 ml) containing LDS-751 solution (10 ml). The samples were mixed again and kept on ice in the dark for 10 min prior to analysis by flow cytometry on a FACSCalibur™ flow cytometer.

Figure 1 shows CDl Ib levels in neutrophils at different chemokine concentrations, and figure 2 shows values for monocytes. The method of the invention is able to provide a 500% increase in detectable signal compared to the baseline (at 1OnM IL-8). For comparison, prior art methods where labelling preceded fixing have seen only a 300% increase using 1OnM IL-8. In parallel experiments, a CXCR2 antagonist (SB-656933) was used in place of the inflammatory chemokines. Whole blood (100 ml) was incubated for 10 min at 37°C with CXCR2 (10 ml) antagonist at 12x final whole blood concentration. A chemokine was added to each sample at 12x final whole blood concentration (10 ml) and incubated for additional 10 min. The tubes were placed on ice and fixative added. 40μl of each sample was added to an Eppendorf tube containing the anti-CDllb and anti-CD16 or anti-CD14 antibodies. The assay then proceeded as described above. The results are shown in Figure 3.

In further experiments, LPS was used to provoke an inflammatory response. Whole blood samples from six healthy volunteers were incubated with LPS at various concentrations, and CDl Ib levels were assayed as described above. A 4-fold increase in signal intensity could be detected.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims

1. A method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of: (i) contacting the substance with a cell; and (ii) assessing the effect of step (i) on cell surface CDl Ib expression by the cell, wherein an increase in CDlIb expression in step (ii) indicates that the substance could cause an in vivo cytokine storm.

2. A method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of: (i) contacting the substance with a cell; (ii) contacting the cell with a fixative; and (iii) contacting the cell with a labelling reagent for CDl Ib, wherein increased CDl Ib labelling in step (iii) indicates that the substance could cause an in vivo cytokine storm.

3. A method for assessing the possible in vivo inflammatory effects of a substance, comprising the steps of: (i) contacting the substance with a cell; (ii) contacting the cell with a fixative; and (iii) contacting the cell with a labelling reagent for CDlIb, wherein the method does not include a step of labelling cell surface markers between steps (i) and (ii).

4. A method for assessing the possible in vivo anti-inflammatory effects of a substance, comprising the steps of: (i) contacting a cell with a potential anti-inflammatory substance; then (ii) contacting the cell with an inflammatory stimulus; and then (iii) assessing the effect of step (i) and (ii) on CDl Ib expression by the cell.

5. A method for assessing the possible in vivo anti-inflammatory effects of a substance, comprising the steps of: (i) contacting a cell with a potential anti-inflammatory substance; then (ii) contacting the cell with an inflammatory stimulus; then (iii) contacting the cell with a CDl Ib fixative; and then (iv) contacting the cell with a labelling reagent for CDl Ib.

6. A method for fixing and labelling a cell surface molecule of a cell, comprising steps of: (i) applying a stimulus to the cell; then (ii) contacting the cell with a surface molecule fixative; and then (iii) contacting the cell with a labelling reagent.

7. A method for fixing surface molecules of a cell, comprising steps of: (i) applying a stimulus to the cell; and then (ii) contacting the cell with a surface molecule fixative, wherein the method does not include a step of labelling cell surface markers between steps (i) and (ii).

8. The method of any preceding claim, wherein the cell in step (i) is a leukocyte.

9. The method of claim 8, wherein the cell in step (i) is a neutrophil.

10. The method of any preceding claim, wherein the cell in step (i) is in a sample of whole blood.

11. The method of any one of claims 2, 3 or 5-10, wherein the fixative is a dilute aqueous solution of formaldehyde.

12. The method of any one of claims 2, 3 or 5-11, wherein the fixative is contacted with the cell within 20 minutes of the end of cell stimulation

13. The method of any one of claims 2, 3 or 5-12, wherein the fixative is contacted with the cell within for 5 minutes or shorter.

14. The method of any one of claims 2, 3 or 5-13, wherein the fixative is used at low temperatures, below room temperature.

15. The method of any one of claims 4-14, wherein the stimulus is a chemical stimulus.

16. The method of claim 15, wherein the stimulus is an antibody or a small organic molecule.

17. The method of any preceding claim, wherein the cell is exposed to a stimulus for up to 1 hour.

18. The method of any preceding claim, wherein cells are labelled with a fluorescently-labelled monoclonal antibody.

19. The method of any preceding claim, wherein cells are labelled with a plurality of labelling reagents, giving different signals from each other, wherein one of the reagents allows different cell types to be distinguished.