method

Abstract

A method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising: a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof; b) exposing the biological sample to a means of determining the cell state; c) contacting the biological sample from b) with dimethylsulfoxide (DMSO) and Formal Saline to fix the cells, cell fragments and / or cell aggregates thereof in the sample; and d) freezing the fixed biological sample.

Description

[0001] METHOD

[0002] The invention relates to a method for fixing and freezing cells, cell fragments and / or cell aggregates thereof in a biological sample obtained from a subject. The fixed and frozen samples may subsequently be thawed and analysed.

[0003] BACKGROUND

[0004] The analysis of cells, cell fragments and / or cell aggregates thereof in a biological sample can be useful for many reasons, for example disease diagnosis, understanding disease progression, monitoring drug efficacy, to name just a few. For example, blood cells including platelets, neutrophils, monocytes, and macrophages must have a normal structure and function to maintain health. By analysing the platelets in a sample of cells, cell fragments and / or cell aggregates thereof from a subject it is possible to identify possible problems. For example, when functioning normally activated platelets aggregate at a site of damage in a blood vessel to form a clot to slow or stop bleeding caused by injury or disease. However, where platelet activation is diminished, bleeding can worsen leading to haemorrhage. Conversely, inappropriate platelet activation can result in blood vessel occlusion.

[0005] Currently, fresh blood samples are needed to assess the structure and function of cells, cell fragments and / or cell aggregates thereof found in blood. This constraint means that such assessments are limited to assays that must be performed quickly after a sample is taken. The assays needed to obtain robust and detailed analyses of the sample, looking at cell structure and function, must typically be performed by trained professionals in specialised facilities. This means that subjects must either attend the specialised facilities, or samples need to be quickly and expensively transported to these facilities. There can also be significant data variability if tests are undertaken at facilities with differing equipment.

[0006] Whilst protocols exist for fixing cells, cell fragments and / or cell aggregates thereof in biological samples derived from a subject, the structure and function of the cell, cell fragment and / or cell aggregate thereof is typically compromised when the fixed samples are cold-stored or frozen (Braathen et al, 2019, Clinical Trial, 59(8): pg. 2652-2661). For example, a reduction in marker specificity may occur during cold or frozen storageof a sample. This is often observed when the marker is a protein probe, such as an antibody, labelled with a fluorescent dye.

[0007] A method for fixing and freezing cells, cell fragments and / or cell aggregates thereof to preserve the ability to measure cellular state (such as structure and function), and thus to allow for accurate, robust, and convenient assessments of cellular structure and function after sample collection and storage, is therefore needed.

[0008] The present invention seeks to address the problems mentioned above. The current inventors surprisingly identified that the method disclosed herein can be used to fix cells, cell fragments and / or cell aggregates thereof in a biological sample obtained from a subject, and that the sample can then be frozen and analysed later e.g., using flow cytometry, and produce results substantially the same as if the sample was analysed before freezing. Analysis of the stored sample can occur at a site, such as a centralised facility, remote in distance and time from where the sample was taken. This thereby avoids the need for specialised equipment or trained staff at the point of sample collection and / or preparation.

[0009] The method allows for high-quality analysis of the sample either immediately after, or days, weeks, months or even years after sample collection, preparation, fixing and freezing. Further, the method does not compromise the binding of a means for determining cell state, for example, antibodies binding to an antigen, nor increase nonspecific binding, nor degrade or enhance the fluorescent properties of fluorophores labelling the antibodies.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention provides a method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising:

[0012] a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof;

[0013] b) exposing the biological sample to a means of determining the cell state; c) contacting the biological sample from b) with one or more fixing reagent to fix the cells, cell fragments and / or cell aggregates thereof in the sample; and

[0014] d) freezing the fixed biological sample.The fixing reagent may comprise DMSO and Formal Saline. The DMSO and Formal Saline may be in the same composition or separate compositions before being contacted with the biological sample.

[0015] Step b) may further comprise exposing the biological sample to a modulator capable of altering the state of the cells, cell fragments and / or cell aggregates thereof.

[0016] In a second aspect, the invention provides a method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising:

[0017] a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof;

[0018] b) contacting the biological sample with DMSO and Formal Saline to fix the cells, cell fragments and / or cell aggregates thereof in the sample; and

[0019] c) freezing the fixed biological sample.

[0020] The biological sample may be blood, serum, urine, a tissue sample, sputum, cerebrospinal fluid etc, anything that can be obtained from the subject that contains cells, cell fragments and / or cell aggregates. More specifically, the biological sample used in the method may comprise platelet-rich plasma (PRP) and / or whole blood. The biological sample used in the method may further comprise platelets, neutrophils, monocytes, macrophages and / or other types of blood cells, cell fragments and / or cell aggregates thereof. The volume of biological sample used in the method may be in a range of about 1 to about lOOpl, preferably in a range of about 3 to about 70pl.

[0021] Further, the biological sample used in the method may also comprise cell aggregates i.e., cells aggregated to other cell types, such as platelet-neutrophil, platelet-monocyte, or platelet-macrophage aggregates. Alternatively, the cells may be aggregated to other biological entities such as viruses or bacteria to form, for example, platelet-bacteria or platelet-virus aggregates or interactions.

[0022] The modulator capable of altering the state of the cells, cell fragments and / or cell aggregates thereof may be a chemical modulator or a physical modulator. A physical modulator may result in a change in temperature in the sample, a change in the physical and shear forces applied to the sample (such as shaking), a change in the pressureapplied to the sample, etc. A chemical modulator may include an inhibitor, antagonist, agonist, or activator. The chemical modulator may be a small molecule, drug, hormone, growth factor, chemokine, cytokine, protein, peptide, nucleic acid, RNA, siRNA, mRNA, small RNA, antisense oligonucleotides, DNA, an aptamer, antibody, or antigen binding fragment.

[0023] More particularly, for platelet analysis the chemical modulator may comprise collagen, adenosine diphosphate (ADP), thrombin, thromboxane A2 (TxA2), matrix metalloproteinases (MMPs) including MMP-1, -2, -3, -9, and / or 14, ristocetin, adrenaline, serotonin, vasopressin, epinephrine, arachidonic acid, peptides derived from agonists such as collagen-related peptide (CRP) or cross-linked variants (CP-XL), thrombin receptor activatory peptides (TRAP) such as thrombin receptor activatory peptide 6 (TRAP-6), or small molecule receptor agonists such as U46619. Inhibitors may include prostacyclin (also called prostaglandin 12 or PGI2), and small molecule analogues may include ralinepag, iloprost, treprostinil, nitric oxide (NO) or NO-donor molecules such as N-[3-aminopropyl(propyl)amino]-N-hydroxynitrous amide (PAPA NONOate) and S-nitroso-N-acetylpenicillamine (SNAP). The chemical modulator may be an anti-platelet drug such as aspirin (or alternate cyclooxygenase [COX] inhibitor), clopidogrel, prasugrel, ticagrelor, ticlopidine (or alternate ADP receptor antagonist), dipyridamole (or alternate adenosine reuptake inhibitor), cilostazol (or alternate phosphodiesterase inhibitor), or vorapaxar (or alternate thrombin receptor- 1 [PAR-1] or -4 [PAR-4] receptor antagonist).

[0024] The chemical modulator may be capable of targeting or cross-reacting with molecules in or on the sample (such as cell signalling molecules, for example, enzymes that control immune function, cell division, migration, or another biological process). The chemical modulator may be ibrutinib, acalabrutinib or fostamatinib.

[0025] The means of determining the cell state may refer to a means that allows the measurement of one or more of:

[0026] • The level of a cell surface marker and / or a change in conformation of a cell surface marker,

[0027] • The level of a secreted protein,

[0028] • The cell size,

[0029] • The cell shape,• The cell number,

[0030] • The degree of cell aggregation,

[0031] • The degree of cell transparency,

[0032] • The degree of cell granularity, or

[0033] • Any other marker which allows the state of the cell, cell fragment or cell aggregate thereof to be assessed.

[0034] Preferably the means of determining the cell state is conjugated to a fluorophore.

[0035] Where the marker of cell state is a cell surface marker, or the change in conformation of a cell surface marker, the means for determining the cell state may be the level of binding of an antibody, antigen binding fragment, or ligand to the cell. The ligand may be a protein, a peptide, an aptamer or a nucleic acid molecule. For example, in the case of the measurement of platelet activation state, the marker of cell state may be the level of cell surface exposure of P-selectin, a transmembrane adhesion receptor secreted to the cell surface; the degree of conformational change in integrin allbp3; or the level of exteriorised membrane phosphatidylserine. P-selectin levels may be determined using a labelled antibody or antigen binding fragment. Conformational changes in integrin allbp3 may be determined directly by antibody binding or antigen binding fragment, or indirectly via fibrinogen binding to the activated integrin and anti-fibrinogen antibody. Phosphatidylserine levels may be determined through binding of labelled annexin V. Cell state may also be determined by using other markers such as an anti-cluster of differentiation 36 (CD36) and / or anti-platelet glycoprotein VI (GPVI) antibody or antigen-binding fragment.

[0036] The cells, cell fragments and / or cell aggregates thereof fixed in step c) of the disclosed method may include cells, cell fragments and / or cell aggregates thereof in a resting / quiescent state, and / or cells, cell fragments and / or cell aggregates thereof in an activated / stimulated state. Exposure of the cells, cell fragments and / or cell aggregates to the modulator may cause the state of at least some of the cells, cell fragments and / or cell aggregates thereof in the biological sample to change.

[0037] The fixing reagents used in the method may include dimethylsulfoxide (DMSO) and / or Formal Saline. Preferably, the fixing reagents include DMSO and Formal Saline. When DMSO and Formal Saline are both used they may be in the same or differentcompositions. When Formal Saline is used in the method the final concentration in step c) in the sample and fix reagent mix, before the sample is frozen, may be in a range of about 0.2 to about 4% by volume, preferably in a range of about 0.3 to about 2% by volume. When DMSO is used in the method the final concentration in step c) in the sample and fix reagent mix, before the sample is frozen, may be in a range of about 2 to about 10% by volume, preferably in a range of about 4 to about 8% by volume. The disclosed method may be methanol-free. Preferably, the method is methanol-free.

[0038] In step d) the fixed cells, cell fragments and / or cell aggregates thereof are frozen. The cells, cell fragments and / or cell aggregates thereof may be frozen at a temperature of at least about -20°C. The cells, cell fragments and / or cell aggregates thereof may be frozen at a temperature of about -80°C. The frozen cells, cell fragments and / or cell aggregates may be stored frozen for at least 1 day, 1 week, 1 month, 3 months, 6 months, 9 months, a year or more before being defrosted / thawed for analysis.

[0039] The method of the invention may be performed in a suitable vessel, for example, a well of a multiwell plate, a vial, a flask, a syringe, an Eppendorf tube, a fluorescence-activated cell sorting (FACS) tube, or other type of suitable vessel.

[0040] Prior to analysis the fixed and frozen samples will be thawed. Some of the DMSO may be removed from the samples prior to analysis, this may be done by centrifuging the sample, removing the majority of the supernatant and resuspending the pellet, for example in a buffer such as Formal Saline, thereby reducing the DMSO concentration.

[0041] The thawed samples containing cells, cell fragments and / or cell aggregates thereof may be analysed using, for example, flow cytometry, FACS, and / or microscopy. In an embodiment, the thawed samples containing cells, cell fragments and / or cell aggregates thereof may be analysed by flow cytometry, and the forward and side scatter observed may allow the size, granularity, count, shape, cycle or other characteristic of the cells, cell fragments and / or cell aggregates thereof in the sample to be determined. Extracts from the sample may be further analysed using, for example, lateral flow, enzyme linked immunosorbent assay (ELISA), and / or quantitative mass spectroscopy.

[0042] Surprisingly the results of the analysis of the samples containing fixed cells, cell fragments and / or cell aggregates thereof before and after freezing are very similar.Preferably, there is little or no statistical difference in the results observed before and after freezing, and this may be in respect of, for example, cell size, cell shape, cell granularity, fluorophore intensity, fluorophore spectral properties and / or marker specificity. Preferably, the results observed after freezing are within 5% or 10% of the results observed before, and this may be in respect of cell size, cell shape, cell granularity, fluorophore intensity, fluorophore spectral properties and / or marker specificity. This is in contrast to known fixing and freezing methods, where after freezing cellular structure and function, fluorophore intensity, fluorophore spectral properties and / or marker specificity are significantly altered.

[0043] EXAMPLES

[0044] Detecting Platelet Activation

[0045] In an embodiment, the method of the invention may be used to study platelet activation. In this embodiment the biological sample may be blood, more specifically it may be platelet-rich plasma (PRP) or platelets isolated from plasma and suspended in a biological buffer solution. The modulator may be an agonist which activates platelets, wherein the agonist may be selected from ADP, CRP or CP-XL, thrombin, TRAP-6, thromboxane A2 (TxA2), matrix metalloproteinases (MMPs) including MMP-1, -2, -3, -9, and / or -14, ristocetin, adrenaline, vasopressin, prostacyclin (PGI2), U46619, epinephrine, serotonin, arachidonic acid and / or collagen. The purpose of the agonist is to modulate the function of the platelets.

[0046] The means of determining the cell state may be an antibody or antigen-binding fragment. Specifically, the antibody or antigen-binding fragment may be used to detect the state (e.g., inherent properties) or function (e.g., change in activity) of platelets and may be selected from an anti-fibrinogen, anti-P-selectin, anti-CD36, and / or anti-GPVI antibody or antigen-binding fragment, or could comprise an antibody or antigen-binding fragment directed to another surface marker to detect the state (e.g., by measuring the levels of cell surface proteins to determine the activation state of platelets) or function of the platelets. Further, the antibody or antigen-binding fragment may be conjugated to a fluorophore. For example, the fluorophores may include Fluorescein isothiocyanate (FIT-C), PE-Cyanine 5 (PE-Cy5), allophycocyanin (APC) and / or Alex Fluor 647.More specifically, the means of determining the cell state may be an anti-fibrinogen and / or anti-P-selectin antibody or antigen-binding fragment labelled with a fluorophore. P-selectin is exposed on the surface of activated platelets following degranulation, and thus is a marker of platelet activation. Fibrinogen binds to activated integrin allbp3 on the surface of activated platelets causing platelet aggregation, thus the presence of fibrinogen is a marker of platelet activation.

[0047] Alternatively, fluorophore-labelled fibrinogen may be used to detect activated platelets, as it will bind to activated integrin allbp3 on the surface of activated platelets.

[0048] Detecting Neutrophil Activation

[0049] In an embodiment, the method of the invention may be used to study neutrophil activation. In this embodiment the biological sample may be whole blood or a fraction of blood, preferably whole blood. The modulator may be an agonist which activates neutrophils, for example the agonist may be N-formyl-methionyl-leucyl-phenylalanine (fMLP), a chemoattractant that activates neutrophils. The means of determining the cell state may be an antibody or antigen-binding fragment. The antibody or antigen-binding fragment may be used to detect activated neutrophils. The antibody or antigen-binding fragment used may be directed to CD 11b, wherein levels of CD 11b are increased on the surface of neutrophils when they are activated. The antibody or antigen-binding fragment may be conjugated to a fluorophore. For example, the fluorophores may include FIT-C, PE-Cy5, APC and / or Alex Fluor 647.

[0050] Detecting Platelet-Neutrophil Interaction

[0051] In an embodiment, the method of the invention may be used to study platelet-neutrophil interaction. During inflammatory responses platelet-neutrophil interactions occur and can be responsible for exacerbating inflammation. The ability of platelets and platelet-mediated factors to trigger neutrophil extracellular trap (NET) formation, and the ability of NETs to trigger platelet function, can result in venous thrombosis. The measurement of platelet-neutrophil aggregate levels is therefore an important clinical measure, although due to the dependence on flow cytometry equipment, specialist expertise and fast sample processes, the use of this measurement is limited. In this embodiment the biological sample may be a blood sample. Measurements may be made in the absence of modulators of this interaction or following exposure to modulators of neutrophils (e g. fMLP) or platelets (e g. ADP, CRP, CRP-XL, thrombin, TRAP-6, TxA2, MMPsincluding MMP-1, -2, -3, -9, and / or -14, ristocetin, adrenaline, vasopressin, prostacyclin (PGI2), PAR-1 and PAR-4 antagonists, U46619, epinephrine, serotonin, arachidonic acid and / or collagen). Levels of platelet-neutrophil aggregation may be measured using platelet- (e.g. CD42b) and / or neutrophil-(e.g. CD14) specific markers. Plateletneutrophil aggregates may be measured using activation state-dependent markers e.g. for platelets, P-selection and / or fibrinogen binding, and for neutrophils, CDllb.

[0052] In a further aspect, the invention provides a method of monitoring drug treatment efficacy and / or safety in a subject comprising:

[0053] a) Providing a first biological sample comprising cells, cell fragments and / or cell aggregates thereof obtained from the subject prior to the administration of treatment;

[0054] b) Performing the method of the first aspect of the invention to produce a fixed and frozen first sample;

[0055] c) Providing a second biological sample comprising cells, cell fragments and / or cell aggregates thereof obtained from the subject during or after the administration of treatment;

[0056] d) Performing the method of the first aspect of the invention to produce a fixed and frozen second sample;

[0057] e) Determining the state of the cells, cell fragments and / or cell aggregates thereof in the samples from steps b) and d); and

[0058] f) Comparing the results determined in step e) to determine the efficacy and / or safety of the treatment.

[0059] The method of steps b) and d) allows the prepared samples to be stored and analysed at the same time.

[0060] The disclosed method of monitoring treatment efficacy and / or safety in a subject may be used a) to screen subjects before, during their or after inclusion in drug trials, b) to test subjects before, during and after exposure to a drug, c) to test the effects of drugs in vitro, d) to test the effects of drugs ex vivo, e) to screen drug libraries to identify on-and off-target effects, and f) as a companion diagnostic to personalise therapeutic strategies.Thus, the disclosed method may be used to identify off-target effects in early-stage clinical trials and monitor efficacy or potential side effects in late-stage clinical trials, thereby minimising risk to the drug development pathway and to patients themselves. It also has the potential for use as a diagnostic or companion diagnostic in clinical practice.

[0061] In another aspect of the invention, there is provided a method of treating a subject who is at risk of a cardiovascular and / or haematological disease or disorder comprising: a) Providing a biological sample obtained from the subject;

[0062] b) Performing the method of the first aspect of the invention to produce a fixed and frozen sample;

[0063] c) Determining the state of the cells, cell fragments and / or cell aggregates thereof in the samples from steps b);

[0064] d) Using the results from c) to determine if the subject is at risk of a cardiovascular and / or haematological disease or disorder; and

[0065] e) Administering to the subject a therapeutic agent to treat or reduce the risk of a cardiovascular and / or haematological disease or disorder if the subject is determined in d) to be at risk of a cardiovascular and / or haematological disease or disorder.

[0066] In another aspect of the invention, there is provided a method of diagnosing abnormal cellular function in a subject, comprising:

[0067] a) Providing a biological sample obtained from the subject;

[0068] b) Performing the method of the first aspect of the invention to produce a fixed and frozen sample;

[0069] c) Determining the state of the cells, cell fragments and / or cell aggregates thereof in the samples from step b);

[0070] d) Using the results from c) to provide a diagnosis to the subject.

[0071] Where the cells, cell fragments and / or cell aggregates thereof are determined by the method of the invention to have high or low levels of cell activation or function measures (such as high or low levels of platelet activation or function measures), this may be indicative that the subject is at increased risk of a cardiovascular and / or haematological disease or disorder. The cardiovascular and / or haematological disease or disorder may comprise bleeding, thrombosis, inflammatory conditions, thrombo-inflammation, infection (e.g., bacterial and / or viral), cancer, and / or autoimmune disease.

[0072] In a yet further aspect, the invention provides a method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising:

[0073] a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof;

[0074] b) contacting the biological sample with DMSO and Formal Saline to fix the cells, cell fragments and / or cell aggregates thereof in the sample; and

[0075] c) freezing the fixed biological sample.

[0076] The “subject” may be a vertebrate, mammal, or domestic mammal. Hence, the method according to the invention may be used to diagnose or treat any animal, for example, pigs, cats, dogs, horses, sheep, or cows. Preferably, the subject is a human.

[0077] The term “treating” or “treatment” refers to the treating or treatment of a disease or medical condition (such as a cardiovascular and / or haematological disorder) in a patient, such as a mammal (particularly a human) which includes: ameliorating the disease or medical condition, i.e. eliminating or causing regression of the disease or medical condition in a patient; suppressing the disease or medical condition, i.e., slowing or arresting the development of the disease or medical condition in a patient; or alleviating the symptoms of the disease or medical condition in a patient. The term encompasses the prophylactic treatment of a disease to prevent or reduce the risk of acquiring or developing a specific disease, or to prevent or reduce the risk of disease recurrence.

[0078] The term “administering”, or “administration”, refers to methods that may be used to enable the delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intranasal, intravitreal, infusion and local injection), transmucosal injection, oral administration, administration as a suppository, and topical administration. One skilled in the art will know of additional methods for administering a therapeutically effective amount of a compound of the present invention for treating, preventing, or relieving oneor more symptoms associated with a disease or disorder such as a cardiovascular and / or haematological disease or disorder.

[0079] By way of example, the volume and / or concentrations of the biological sample, modulator (e.g., agonist), means of determining the cell state (e.g., antibody), reagent, and / or buffer used may be higher (or increased by) at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more compared to the volumes or concentrations recited herein. Alternatively, the volume and / or concentrations of the biological sample, modulator, means of determining the cell state, reagent, and / or buffer used may be lower by (or reduced by at) least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more compared to the volumes or concentrations recited herein.

[0080] By way of example, the volume and / or concentrations of the biological sample, modulator, means of determining the cell state, reagent, or buffer used may be at least 2, 5, 10, 15, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000, or more-fold higher than the volumes or concentrations recited herein. By way of example, the volume and / or concentrations of the biological sample, modulator, means of determining the cell state, reagent, or buffer used may be at least 2, 5, 10, 15, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000, or more-fold lower than the volumes or concentrations recited herein.

[0081] All of the embodiments and features described herein (including any accompanying claims, abstract and drawings) and / or all of the steps of any method or process as disclosed may be combined with any of the above aspects or embodiments in any combination, unless stated otherwise or where at least some or such features and / or steps are mutually exclusive.

[0082] BRIEF DESCRIPTION OF THE DRAWINGS

[0083] There now follows, by way of example only, a detailed description of the present invention with reference to the accompanying drawings, in which:Figure 1 shows that platelet capacity (the scale or size of the response) is preserved between a) platelets activated, fixed, and then frozen using the disclosed method and b) platelets activated and then fixed but not frozen. Cellular agonists ADP, CRP and TRAP-6 were used to activate platelets, and two markers were used to assess platelet activation: fibrinogen binding to the platelet surface and P-selection exposure on the platelet surface. Each dot represents an individual donor, and each parameter is derived from a full concentration-response curve.

[0084] Figure 2 shows that platelet sensitivity (the activator concentration that stimulates half-maximal response, or effective concentration 50 (ECso)) is preserved between a) platelets activated, fixed, and then frozen using the disclosed method and b) platelets activated and then fixed but not frozen. Again, ADP, CRP and TRAP-6 were used to activate platelets, and fibrinogen binding to the platelet surface and P-selection exposure on the platelet surface was assessed to evaluate platelet activation. As in Figure 1, each dot represents an individual donor, and each parameter is derived from a full concentration-response curve.

[0085] EXAMPLES

[0086] Example 1

[0087] Plate preparation

[0088] To assess platelet function, two 96-well Platelet Phenomics Analysis (PPA) plates were prepared. Plates were first coated with gelatine by incubation for 4 hours in a 0.75% (w / v) solution in phosphate buffer containing 0.05% (v / v) Tween-20. Plates were then washed in 0.01% (v / v) Triton X-100 containing double-distilled water (ddELO), before double washing in ddELO and air drying. Concentration ranges of platelet activators were then added to separate wells on each plate (lOpL: ADP, TRAP-6 or CRP, each dissolved in water containing human albumin and ascorbic acid) pre-determined to produce a full range of platelet function outcomes, from no response (no activator) to a maximum response (maximum activator concentration). Plates were freeze dried overnight at -60°C and then vacuum sealed for storage. Prior to use, the reagents in each well were rehydrated with 25 pL HEPES-buffered saline.

[0089] A Mastermix was prepared including fluorescently labelled antibodies (specific for P-selectin - a marker of platelet degranulation, and fibrinogen - a protein that binds to activated platelets), additional HEPES Buffered Saline (HBS) and platelet-rich plasma(PRP, the fraction of blood plasma obtained following centrifugation of whole human blood at 100 x g for 20 minutes), as shown in Table 1. lOpl of the Mastermix was then added into each test well in both plates.

[0090] Table 1

[0091]

[0092] The plate containing the rehydrated agonist and the Mastermix (comprising PRP, HBS, agonists and antibodies) was then incubated at room temperature for 20 minutes in the dark.

[0093] Experiment-appropriate controls (such as FITC-fibrinogen-negative controls and PE-Cy5 P-selectin-negative controls) were added to other wells of the plate that did not contain agonists, as required.

[0094] Cryopreservation of plate

[0095] After incubation, the cells were prepared for assay freezing as follows:

[0096] 140pl of fix-freeze reagent comprising: 1% Formal Saline (1% Formaldehyde methanol free in 137mM NaCl, 2.7mM KC1 and 10 mM phosphate buffer solution (pH 7.4 at 25°C)), and 6.86% DMSO was added to each well. After the addition of the fix-freeze reagent into each well already containing 20pl of sample, the final concentration of fixfreeze reagents in each well comprised 0.875% Formal Saline and 6% DMSO.

[0097] Each well was then mixed, following which one plate was sealed and stored at -80°C for 1 month and the other plate was analysed immediately.

[0098] Analysis of plate

[0099] After a month, the frozen plate was then taken out of the -80°C freezer and left at room temperature to defrost. After defrosting, the DMSO concentration was reduced by centrifuging the plate at 1413g for 10 minutes at 20°C, with brake applied. 140pl of supernatant was then removed without disturbing the pellet.The remaining sample was then resuspended in 140pl of 0.2% Formal Saline (comprising 0.2% Formaldehyde methanol-free in 137mM NaCl, 2.7mM KC1 and ImM phosphate buffer solution (pH 7.4 at 25°C)) to yield a final concentration of 0.19% Formal Saline.

[0100] Flow cytometry was then used to assess the levels of fluorescent signal, which corresponds to the amount of labelled antibodies bound to the cells. In this case, flow cytometry was used to establish the levels of fibrinogen binding to platelets and P-selectin exposure on the platelet surface to enable the quantification of platelet function properties, for example the sensitivity of platelets to an agonist or the scale of the response to such an agonist.

[0101] For non-frozen samples, the plate was analysed using flow cytometry within an hour of cellular fixation following application of the fixing reagents.

[0102] Results

[0103] The results obtained using flow cytometry showed that activated platelets before and after fixing and freezing using the method of the invention presented with highly conserved capacity and sensitivity characteristics.

[0104] Specifically, both platelet capacity as shown in Figure 1 (wherein capacity refers to the scale of response to each cellular agonist used) and platelet sensitivity as shown in Figure 2 (wherein sensitivity refers to the activator concentration that stimulates half-maximal response, or ECso for each cellular agonist used) was found to be highly correlated between individual donors for platelets that had been activated, fixed, and then frozen using the disclosed method compared with platelets that were activated and fixed but not then frozen.

[0105] These results indicate the effectiveness of the method of the invention.

[0106] Example 2

[0107] Analysis of platelets using alternative concentrations of fix-freeze reagent

[0108] As a separate example, in similar experiments (using the same methodology as outlined in Example 1) fibrinogen binding to platelets was assessed following stimulation withagonists ADP, CRP or TRAP-6 and the treatment of 20pl samples with 140 pl fix-freeze reagent comprising 0.5% Formal Saline and 6.86% DMSO (giving final concentrations of 0.43% Formal Saline and 6% DMSO).

[0109] Results

[0110] Platelet capacity and sensitivity outcomes from two donors were analysed and shown to be highly similar when outcomes were analysed before freezing the samples and then 1 month after freezing the samples, as shown in Table 2.

[0111] Table 2

[0112]

[0113] These results again indicate the effectiveness of the method of the invention using a range of concentrations of fix-freeze reagent.

[0114] REFERENCES

[0115] 1. Braathen H, Sivertsen J, Lunde THF, Kristoffersen EK, Assmus J, Hervig TA, Strandenes G, Apelseth TO. In vitro quality and platelet function of cold and delayed cold storage of apheresis platelet concentrates in platelet additive solution for 21 days. Transfusion. 2019 Aug;59(8):2652-2661. doi: 10.1111 / trf.15356.

Claims

CLAIMS1. A method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising:a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof;b) exposing the biological sample to a means of determining the cell state; c) contacting the biological sample from b) with dimethylsulfoxide (DMSO) and Formal Saline to fix the cells, cell fragments and / or cell aggregates thereof in the sample; andd) freezing the fixed biological sample.

2. The method of claim 1, wherein the biological sample from b) is also contacted with a modulator capable of altering the state of the cells, cell fragments and / or cell aggregates thereof.

3. The method of claim 1 or 2, wherein the method is methanol-free.

4. The method of any preceding claim, wherein the final concentration of Formal Saline in the fixed sample is in a range of about 0.2 to about 4% by volume, optionally in a range of about 0.3 to about 2% by volume.

5. The method of any preceding claim, wherein the final concentration of DMSO used in the fixed sample is in a range of about 2 to about 10% by volume, optionally in a range of about 4 to about 8% by volume.

6. The method of any preceding claim, wherein the biological sample is plateletrich plasma (PRP) and / or whole blood.

7. The method of claim 6, wherein the biological sample comprises platelets, neutrophils, monocytes, macrophages and / or other types of cells, cell fragments and / or cell aggregates thereof.

8. The method of claim 6, wherein the volume of PRP used is in a range of about 1 to about lOOpl, optionally in a range of about 3 to about 70pl.

9. The method of claim 2, wherein the modulator is selected from acalabrutinib, adenosine diphosphate (ADP), adrenaline, arachidonic acid, aspirin, cilostazol, clopidogrel, collagen, collagen-related peptide (CRP), cross-linked collagen-related peptide (CRP-XL), dipyridamole, epinephrine, fostamatinib, N-formyl-methionyl-leucyl-phenylalanine (fMLP), ibrutinib, iloprost, metalloproteinase (MMP)-l, -2, -3, - 9. and / or -14, nitric oxide (NO), N-[3-aminopropyl(propyl)amino]-N-hydroxynitrous amide (PAPA NONOate), prasugrel, prostacyclin (PGI2), ralinepag, ristocetin, serotonin, S-nitroso-N-acetylpenicillamine (SNAP), thrombin, thrombin receptor activatory peptide (TRAP)-6, , thromboxane A2 (TxA2), ticagrelor, ticlopidine, treprostinil, U46619, and / or vorapaxar.

10. The method of any preceding claim, wherein the means of determining the cell state is an antibody or antigen-binding fragment.

11. The method of claim 10, wherein the antibody or antigen-binding fragment is one or more of anti-cluster of differentiation 36 (CD36), 11b (CDl lb), 42b (CD42b) and / or 14 (CD 14), anti-fibrinogen, anti -platelet glycoprotein VI (GPVI) and / or anti-P-selectin antibody or antigen-binding fragment.

12. The method of claim 10 or 11, wherein the antibody or antigen-binding fragment is conjugated to a fluorophore, optionally wherein the fluorophore is Fluorescein isothiocyanate (FIT-C), PE-Cyanine 5 (PE-Cy5), allophycocyanin (APC) and / or Alex Fluor 647.

13. The method of any preceding claim, wherein steps b) and c) are performed in a vessel, optionally wherein the vessel is a multiwell plate.

14. The method of any preceding claim, further comprising the step of thawing and analysing the fixed and frozen sample comprising cells, cell fragments and / or cell aggregates thereof.

15. A method of fixing and freezing cells, cell fragments and / or cell aggregates thereof, the method comprising:a) providing a biological sample obtained from a subject wherein the sample comprises cells, cell fragments and / or cell aggregates thereof;b) contacting the biological sample with dimethylsulfoxide (DMSO) and formal saline; andc) freezing the fixed biological sample.

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