WO2025012440A1

WO2025012440A1 - Method for detection of the potential of a body fluid sample to produce extracellular traps

Info

Publication number: WO2025012440A1
Application number: PCT/EP2024/069850
Authority: WO
Inventors: Jacob Vincent Micallef
Original assignee: Belgian Volition SPRL
Current assignee: Belgian Volition SPRL
Priority date: 2023-07-13
Filing date: 2024-07-12
Publication date: 2025-01-16
Anticipated expiration: 2026-01-13

Abstract

The invention relates to methods for the detecting or measuring of the potential of cells in a body fluid to produce extracellular trap material which comprises comparing the level of extracellular traps (ETs) measured in a first and second sample of the body fluid, wherein the second sample is incubated for NETosis to proceed for a longer period of time than the first sample. The invention also relates to methods of diagnosing and monitoring disease by investigating the propensity of a body fluid sample obtained from the subject to produce ETs and/or NETs using the methods described.

Description

METHOD FOR DETECTION OF THE POTENTIAL OF A BODY FLUID SAMPLE TO PRODUCE EXTRACELLULAR TRAPS

FIELD OF THE INVENTION

The invention relates to methods of detecting and measuring the potential of a biological sample to produce extracellular traps, including neutrophil extracellular traps. Such methods allow for improved detection and diagnosis of diseases associated with dysregulated extracellular trap formation.

BACKGROUND OF THE INVENTION

White blood cells are an important component of the innate immune system. They are recruited to the site of an infection as part of the immune response where they protect against a broad range of pathogens by mechanisms including degranulation, phagocytosis and intracellular degradation of pathogens as well as by the formation of extracellular traps (ETs). Neutrophils make up some 60% of human white blood cells and can form neutrophil extracellular traps (NETs). NETs are the best known and most studied ETs. ETs and NETs are web-like decondensed chromatin structures ejected extracellularly by white blood cells, particularly activated neutrophils, as a natural mechanism to trap pathogens such as bacteria, viruses, fungal spores and parasites. ETs and NETs help to prevent infection by trapping and degrading invading pathogens locally and also help prevent their spread throughout the body.

Huge numbers of neutrophils are generated daily in the bone marrow from myeloid progenitor cells. The myeloid cells progress through a number of neutrophil precursor cells, including myeloblasts, promyelocytes, myelocytes, metamyelocytes and band cells before becoming mature neutrophils. Under normal conditions, neutrophils are thought to live less than 24 hours after entering the circulation. During this time neutrophils take on various phenotypes, including activated phenotypes, as they migrate out of the vasculature into the tissues where they complete their functions before being eliminated by macrophages. However, some neutrophils may persist for longer times including some extravasated neutrophils.

Neutrophil biology is not well understood and remains a subject of study. Neutrophils are highly plastic and heterogeneous in phenotype and function. The neutrophil phenotype and activation state may change with age at different points in the neutrophil life-history. Neutrophils are abundant in the blood but also occur in many different tissues through extravasation from the vascular system, for example into the lungs or spleen. Neutrophils located in different tissues may have different phenotypes representing different neutrophil sub-populations. Different neutrophil phenotypes and different activation states may occur in different disease states, for example in infection or inflammation. Neutrophils are also thought to have a variety of functions not limited to immunity.

Neutrophils migrate to the site of an injury, infection, inflammation, or other insult to the body as a response to chemotaxis. Thus, neutrophils may be found in many different tissues and in many body fluids if recruited there in response to an infection or injury. Neutrophils have been observed in bronchoalveolar lavage fluid (BALF), cerebrospinal fluid (CSF), sputum, saliva, urine and stool.

The ejection of decondensed chromatin to produce NETs by neutrophils involves a process known as NETosis. NETosis of a neutrophil may be stimulated or induced by many different factors including by the presence of a bacterial, fungal or viral pathogen, as well as by many chemical activators including, without limitation, heparin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), reactive oxygen species such as hydrogen peroxide or moieties that generate reactive oxygen species (for example, glucose oxidase), ionomycin or calcium ionophores (Cal) (Fuchs et al, 2007, Neubert et al, 2019).

NETosis may also be induced by the complement system and coagulation of blood. Coagulation or clotting of blood at an open wound stimulates NETosis providing a scaffold on which platelets and blood cells may aggregate during clot formation to close the wound, whilst also providing protection against infection by trapping and degrading any pathogens that may enter the body locally and thus preventing the spread of infection to other parts of the body.

The process of NETosis is well known in the art and described in reviews (Fousert et al, 2020, Thalin etal, 2019, Sollberger ef al, 2018, Snoderly etal, 2019, Niedzwiedzka- Ryswej et al, 2019, Papayannopoulos, 2018, Delgado-Rizo et al, 2017). NETosis involves decondensation of tightly coiled chromatin in the nucleus of a neutrophil to produce unwound strings of chromatin with a concomitant increase in nuclear size in response to intracellular mediators that include reactive oxygen species, produced by NADPH oxidase or mitochondria, which activate myeloperoxidase (MPO), neutrophil elastase (NE) and protein-arginine deiminase type 4 (PAD4). The decondensed chromatin is ejected extracellularly to form NETs. Pathogens are trapped and subjected to degradation by histone and DNA components of NETs which have antimicrobial properties. NETs also contain additional antimicrobial moieties to neutralize captured pathogens including for example myeloperoxidase and proteolytic enzymes.

Once produced by NETosis, NETs are reported to persist for some 24 hours in the vasculature (Kolaczkowska et al, 2015), but are eventually degraded by mechanisms thought to include digestion of DNA by DNase enzymes secreted into the circulation and by phagocytosis (Farrera and Fadeel, 2013).

Activated neutrophils may undergo NETosis if induced to do so, for example by the presence of a pathogen. Additionally, neutrophils secrete a variety of pro-inflammatory cytokines and surface molecules (major histocompatibility complex II) leading to altered membrane composition and increased activity in the cytoplasm (for example to produce cytokines). Excessive cytokine release may lead to further neutrophil activation, NETosis and, in turn, further cytokine release in a classic “cytokine storm” positive feedback loop, for example as has been associated with severe sepsis and COVID-19.

Neutrophils in circulation also release harmful intracellular granule contents when recruited, primed, and activated following encounters with chemokines, cytokines or pathogens. However, neutrophils can also become primed or activated during immune dysregulated conditions such as sepsis where primed neutrophils exhibit a 10 to 20- fold increase in their response if stimulated to do so. Septic patients have been described to contain a primed population of neutrophils. Similarly, acute respiratory distress syndrome (ARDS) patients are reported to have high plasma levels of tumor necrosis factor-a (TN Fa) which primes neutrophils resulting in hyper-responsiveness and lung injury. Overactivation of neutrophils may result in dysfunction and cause tissue damage. For example, while neutrophil recruitment in response to lung injury is an appropriate response, excessive recruitment of activated neutrophils into the pulmonary vasculature may lead to ARDS in patients with severe trauma or hemorrhagic shock.

Whilst NETs are very effective in protection against infection by trapping invading pathogens, excessive NETosis is a major cause of pathology and is involved in a large number of disease processes including, without limitation, sepsis, metabolic diseases, cancer, obesity, most or all autoimmune conditions, most or all inflammatory conditions, Alzheimer’s disease, atherosclerosis, bacterial infection, cystic fibrosis, pancreatitis, viral infection, type I and type II diabetes, cancer, vasculitis, thrombosis, pneumonia, respiratory infections, gout, rheumatoid arthritis (RA), psoriasis, systemic lupus erythematosus, atherosclerosis, stroke and sickle cell disease (Sollberger et al, 2018, Thalin et al, 2019, Neubert et al, 2019). The list of diseases in which NETs plays a pathological role is a growing list as workers discover NETs association in more diseases.

Inappropriate production of NETs is not only associated with these diseases but is a causative factor in disease mechanism, disease progression or disease severity. The prolonged presence of NETs may cause tissue damage and development of an autoimmune reaction against NETs components leading to inflammatory, autoimmune, and vascular diseases. Cytotoxic proteases and histones in NETs may cause endothelial damage in sepsis and small vessel vasculitis. In severe influenza, the alveolar-capillary surfaces of the lungs may become embroiled with NETs and damaged by cytotoxic NETs-associated proteins including histones and MPO (Moorthy et al, 2013). Highly elevated plasma NETs levels have been observed in patients with COVID-19 and sepsis. In hospitalised COVID-19 and sepsis patients, plasma NETs levels are higher in patients with severe disease (with organ failure), than in patients with less severe disease. Plasma NETs levels also correlate with disease severity as assessed by Sequential Organ Failure Assessment (SOFA) Score and are predictive of mortality (Morimont et al, 2022, Rea et al, 2021). NETs measurements in bronchoalveolar lavage fluid samples taken from patients with pneumonia (Maruchi et al, 2018) and in serum samples taken from patients with COVID-19 infections (Zuo et al, 2020) have shown that NETs levels were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. NETs levels therefore predict which patients are in need of high levels of respiratory support.

NETs are implicated as a cause of cancer related thrombosis and as a facilitator of metastatic stage IV cancer disease progression by a variety of mechanisms including through the entrapment of tumour cells in NETs, facilitating the spread of NET-bound tumour cells around the body and by assisting in the establishment of metastatic cell growth at new locations (Teijeira et al, 2020, Rayes et al, 2019, Cools-Lartigue et al, 2013, Niedzwiedzka-Ryswej et al, 2019). Rayes et al, 2019 reported that the level of circulating NETs measured in heparin plasma samples is elevated in late stage cancer and correlates with disease stage. They also reported that metastatic disease progression is inhibited by inhibition of NETs formation and that the level of circulating NETs is a better predictor of tumor progression than neutrophil count or neutrophil to lymphocyte ratio.

As ETs and NETs material consists primarily of long strings of nucleosomes connected by DNA and also contains MPO and NE, the level of NETs can be detected by measuring the level of circulating cell free DNA (cfDNA), cell free nucleosomes (including citrullinated cell free nucleosomes), MPO, NE, cell free MPO-DNA complexes or cell free NE-DNA complexes (Thalin et al, 2019). As NETs are reported to be citrullinated, the level of histones or citrullinated histones may also be measured.

The literature reports three main types of measurements relating to NETs and/or NETosis in blood samples. The first type of NETs measurement relates to the simple measurement of the level of NETs in a blood sample (i.e. the actual level of NETs present in the circulation at the time of sampling). This can be measured, for example, as the level of circulating cell free nucleosomes in a subject.

The second type of measurement involves the culturing of neutrophils isolated from a blood sample taken from a subject and measuring the activation state of the subject’s neutrophils in a functional test of NETosis in vitro, typically triggered or induced using a drug such as PMA or ionomycin. The neutrophil cells are assessed for their activation state or tendency to undergo NETosis in cell culture using various cell-based assays and techniques. The capacity of neutrophil cells isolated from patients to undergo NETosis in cell culture varies with physiological states indicating that diverse neutrophil subpopulations are clinically relevant in a wide variety of NETs associated disease states (Rosales, 2018). Neutrophil activation of isolated neutrophils has been assessed in cell culture medium by multiple workers in multiple disease areas. For example, Rayes et al, 2019 showed that neutrophils taken from tumor bearing mice were more sensitive to induction of NETosis using PMA than neutrophils taken from control mice with no tumor. NETosis was measured as the increase in the size of neutrophil nuclei produced on chromatin condensation. As the neutrophils used in the experiment were isolated and cultured in cell culture media, Rayes et al, 2019 concluded that the difference observed in cancer was related to the neutrophil cells and that primary tumours prime neutrophils to release NETs. Demers et al, 2012 reported that peripheral blood neutrophils isolated from leukaemic and mammary and lung cancer bearing mice are more prone to NET formation ex vivo. Moreover, this elevated tendency to NETosis contributes to thrombosis. Similarly, Wolach et al, 2018 isolated and cultured neutrophils from patients with myeloproliferative neoplasms and myelodysplastic syndrome. They reported increased NET formation in neutrophils isolated from patients with myeloproliferative neoplasms compared to age-matched controls or patients with myelodysplastic syndrome. Wong et al, 2015 showed that neutrophils in cell culture isolated from individuals with either type 1 or type 2 diabetes were more susceptible to NETosis when stimulated with the calcium ionophore, ionomycin or LPS than neutrophils taken from control subjects. Wong et al, 2015 measured NETosis as the proportion of isolated neutrophils that visibly produced NETs under microscopy and by Western blot analysis of neutrophil citrullinated histone H3 content or peptidylarginine deiminase 4 (PAD4) content. They concluded that diabetes primes neutrophils to undergo NETosis which severely impairs wound healing resulting in significant diabetic morbidity and mortality. Therefore, measurements of neutrophil propensity are useful in diabetes for the prediction and risk stratification of patients who may develop impaired would healing, vascular and micro-vascular disease, and other complications of diabetes including foot ulcers and amputation. Park et al, 2017 reported a similar effect in sepsis patients where neutrophils in cell culture isolated from patients with sepsis showed an increased response to PMA stimulation and the level of stimulation was predictive of patient survival. In this case NETosis was measured by adding a fluorescent DNA stain to the isolated neutrophil cells to quantify the DNA produced by NETosis. They concluded that neutrophils isolated from patients who had survived sepsis were primed for NET formation in response to subsequent PMA stimulation. In another example, Barbu et al, 2019 showed that neutrophils isolated from patients with sickle cell disease are primed towards NETs production, have a NETotic phenotype and produce more NETs relative to neutrophils taken from healthy subjects. Overall, this second type of measurement relates to neutrophil activation and the potential of isolated neutrophil cells to produce NETs, if triggered to do so. However, the investigation of a subject’s neutrophils in cell culture is not a practical method for routine rapid, high throughput clinical testing.

A slightly different example of a cell culture-based assay was described by Zuo et al, 2020 who isolated neutrophil cells from healthy subjects, cultured them in RPMI cell culture medium, and used them to investigate whether the serum of patients diagnosed with a COVID-19 infection could act as a stimulant or inducer of NETosis. They reported that addition of diluted serum samples taken from COVID-19 patients triggered NETosis in cultured neutrophils from healthy subjects more robustly than heterologous control sera.

A third type of NETs measurement described in the literature is a functional test for the amount of NETs produced by neutrophils in a whole blood sample, when triggered to do so. One feature of this method is that the potential of neutrophils in the blood sample for NETosis is determined in the blood sample itself (i.e. in the natural environment of the neutrophils) rather than in cell culture media. As NETosis may be induced by coagulation, collection of whole blood in a serum blood collection tube (BCT) leads to spontaneous ex vivo NETosis. The level of NETs induced ex vivo in a whole blood sample outside of the body may therefore be measured without the use of drugs to induce NETosis. Sur Chowdhury et al, 2014 reported a method in which a whole blood sample was taken into a serum BCT and allowed to clot for 1 hour (± 5 minutes) before separating the serum from the cells by centrifugation and removing the supernatant serum. Measurement of the amount of NETs induced by clotting for 1 hour was reported to be a better marker for rheumatoid arthritis (RA), with a higher Area Under the Curve (AUC) of 0.97, than the actual level of in vivo circulating NETs measured in ethylenediamine tetraacetic acid (EDTA) plasma with an AUC of 0.57 (i.e. as measured by the first type of NETs measurement described above). Sur Chowdhury et al, 2014 attributed the observed higher level of ex vivo NETs production in RA patients, compared to healthy subjects, to enhanced signalling elements associated with NETosis in RA patients. These test methods would be amenable for use in routine patient care but have never been adopted. We show herein, that these methods do not produce optimal results and are not reliable.

Ebrahimi et al, 2018 reported that elevated serum levels of NETs are associated with increased mortality in community acquired pneumonia. Unlike Sur Chowdhury et al, 2014, however, the method of collection of the serum samples involved blood draw followed by immediate centrifugation. As collection of serum samples normally requires coagulation of the whole blood for 20-30 minutes prior to centrifugation, the sample collection method used in these studies is not clear. In addition, immediate centrifugation would leave no time for NETosis to proceed. This third type of measurement relates to neutrophil activation and the potential of neutrophil cells in blood to produce NETs, if triggered to do so by coagulation.

NETs and NETosis are subject to a great deal of research interest and are clearly of clinical relevance in many disease areas. It is clear that NETs and NETosis are associated with and/or a causative factor in a wide variety of disease processes. The propensity of neutrophils to NETosis is an extremely useful monitor of the health or state of the immune system of a subject. The measurement of the propensity for NETosis, particularly a propensity or pre-disposition to dysregulated or excessive NET osis, has a very wide array of applications including, for example without limitation, diagnostic and prognostic applications for the risk of severity of a broad range of diseases from rheumatoid arthritis to cancer to sepsis, and to determine and monitor the efficacy of immune related or other therapies and/or their effect on the immune system. Despite this, no functional tests for neutrophil activation are used in patient care. This is because such tests are either performed in cell culture or do not perform well.

Although no functional tests of the immune system are in routine clinical use, some tests to detect activated neutrophil structure subtypes by fluorescence activated cell sorting are commercially available, for example the Sysmex Corporation NEUT-GI and NEUT-RI tests.

A reliable, functional test for the propensity of white blood cells to undergo NETosis would be useful for the diagnosis and management of many patients in many disease areas, in particular to identify patients at high risk for excessive or dysregulated NETosis or with a high capacity or propensity for NETosis.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a method for the detection or measurement of the potential of cells in a body fluid to produce extracellular trap material which comprises comparing the level of extracellular traps (ETs) measured in a first and second sample of the body fluid, wherein the second sample is incubated for a longer period of time than the first sample.

According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of cells in a body fluid to produce extracellular trap material wherein said method comprises the steps of:

(i) measuring the level of extracellular traps (ETs) in a first sample of the body fluid;

(ii) incubating a second sample of the body fluid, optionally in the presence of a moiety to trigger NETosis;

(iii) measuring the level of ETs in the second sample; and (iv) using the difference in the measured levels of ETs in the first and second samples as an indicator of the potential of cells in the body fluid, to produce extracellular trap material.

According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs, wherein said method comprises the steps of:

(i) measuring the level of ETs in a first whole blood sample obtained from the subject, or in serum or plasma derived from the first whole blood sample;

(ii) incubating a second whole blood sample obtained from the subject to allow NETosis to occur;

(iii) measuring the level of ETs in the second whole blood sample, or in serum or plasma derived from the second whole blood sample; and

(iv) using the difference in the measured levels of ETs in the first and second samples as a measurement of the potential of the whole blood sample obtained from the subject to produce ETs.

According to a further aspect of the invention there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs, wherein said method comprises the steps of:

(i) incubating a first whole blood sample, optionally with agitation of the sample, to allow coagulation to occur, wherein the first whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel;

(ii) processing the first whole blood sample to terminate NETosis and/or to isolate serum from the sample, and measuring the level of ETs;

(iii) incubating a second whole blood sample for a longer time than the first sample, optionally with agitation of the sample, to allow NETosis to occur, wherein the second whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel;

(iv) processing the second whole blood sample to terminate NETosis and/or to isolate serum from the sample, and measuring the level of ETs; and

(v) using the difference in the measured levels of ETs in the first and second samples as a measurement of the potential of the whole blood sample obtained from the subject to produce ETs. According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a body fluid obtained from a subject to produce NETs, wherein said method comprises the steps of:

(i) measuring a level of NETs in a first sample of the body fluid;

(ii) adding an inducer of NETosis to a second sample of the body fluid;

(iii) incubating the second sample for the development of a measurable level of NETs;

(iv) centrifuging the second sample;

(v) isolating the supernatant component of the second sample;

(vi) measuring the level of NETs in the supernatant of the second sample; and

(vii) using the difference in the levels of NETs measured in steps (i) and (vi) as an indicator of the potential of the body fluid sample to produce NETs.

According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce coagulation induced ETs, wherein said method comprises the steps of:

(i) measuring the level of ETs in a first whole blood sample, or in serum derived from the first whole blood sample, wherein the first whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel;

(ii) incubating a second whole blood sample to allow NETosis to occur, wherein the second whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel;

(iii) measuring the level of ETs in the second whole blood sample, or in serum derived from the second whole blood sample; and

(iv) using the difference in the measured levels of ETs in steps (i) and (iii) as a measurement of the potential of the whole blood sample obtained from the subject to produce coagulation induced ETs.

According to a further aspect of the invention, there is provided a method for the diagnosis, prognosis or monitoring of an actual or suspected disease state or syndrome associated with dysregulated or elevated levels of ETs and/or NETs in subject using a method of the invention. According to a further aspect of the invention, there is provided a method for assessing the anti-inflammatory effect of a therapy in a subject comprising the steps of:

(i) administering the therapy to the subject;

(ii) investigating the propensity of a body fluid sample obtained from the subject to produce ETs and/or NETs using a method according to the invention, to assess the inflammatory status of the subject on one or more occasions;

(iii) using the results obtained in step (ii) to assess the anti-inflammatory effect of the therapy.

According to a further aspect of the invention, there is provided a method of treating a subject diagnosed with, or suspected of, a disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs, comprising the steps of:

(i) investigating the propensity of a whole blood sample obtained from the subject to produce ETs and/or NETs by a method according to the invention;

(ii) using the results obtained in step (i) to determine the treatment required for the subject; and

(iii) administering the treatment to the subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The 24 hour (A) and 4 day (B) time course of nucleosome release in whole blood collected in serum tubes. The level of NETs produced in whole blood collected from 4 (Fig. 1A) or 5 (Fig. 1 B) healthy volunteers in serum blood collection tubes and centrifuged at various times up to 96 hours following venepuncture was measured as the level of H3.1 -nucleosomes. The figure shows that nucleosomes and NETs were generated continuously in whole blood and the level observed increases with time.

Figure 2. Bioanalyzer analysis of cfDNA present in EDTA plasma and serum samples left to produce NETs for 96 hours. Bioanalyzer traces were generated for (A) the background cell free chromatin fragments present in samples taken from 2 healthy volunteers in EDTA plasma, or for (B) serum samples where the whole blood was left for NETosis to occur for 4 days prior to centrifugation. The majority of cfDNA in EDTA plasma lies in a single peak comprising small DNA fragments of approximately 170 base pairs (bp) in length which is consistent with a size expected for mono-nucleosomes. There is also a smaller di-nucleosome peak at approximately 340bp (see Fig. 2A). In contrast, there is much more cfDNA in serum tubes left for NETosis to occur and this comprises mono-nucleosome and di-nucleosome peaks as well as a tri-nucleosome peak at approximately 510bp and a large quantity of bigger cfDNA fragments with sizes ranging from 700 to more than 10,000 bp representative of large chromatin fragments containing many nucleosomes (see Fig. 2B). These results show that the chromatin fragments produced by NETosis increase over time.

Figure 3. NETosis does not occur in whole blood collected in EDTA plasma tubes. The level of NETs produced in whole blood collected from 20 healthy volunteers in EDTA plasma tubes and centrifuged at various times up to 24 hours following venepuncture was measured as the level of H3.1 -nucleosomes. No increase in nucleosomes was observed in 24 hours for 19 of the 20 subjects.

Figure 4. NETosis does not occur in whole blood collected in STRECK cell free DNA plasma blood collection tubes. The level of NETs produced in whole blood collected from 9 healthy volunteers in Streck cfDNA BCT plasma tubes and centrifuged at various times following venepuncture was measured as the level of H3.1- nucleosomes. No increase in nucleosomes was observed between 2-24 hours.

Figure 5. The level of circulating nucleosomes measured in EDTA plasma samples. EDTA plasma samples were taken from 5 healthy volunteers as well as 10 subjects diagnosed with Diabetes Mellitus Type I or Type II and 2 subjects diagnosed with Rheumatoid Arthritis (RA). The level of circulating nucleosomes was measured as an indicator of the amount of NETs and NETs metabolites present in the circulation of patients. The mean level of circulating nucleosomes observed in diabetic patients (52mg/ml) was higher than that observed for healthy subjects (39ng/ml). However, the range of levels in healthy subjects was such that only 3 of 10 diabetic patients had levels higher than the highest level observed for any the healthy volunteers. Similarly, 1 of the 2 RA patients had elevated levels of circulating nucleosomes, whilst the second patient had very low levels.

Figure 6. The level of NETs generated in whole blood in serum blood collection tubes with no mixing. The level of NETs generated in the same 17 subjects described for Figure 5, was measured as: (A) the level of NETs generated in whole blood left standing in a test tube rack for 20 minutes with no mixing, measured as the H3.1 -nucleosome level in serum separated from the whole blood by centrifugation 20 minutes post venepuncture; (B) the level of NETs generated in whole blood left standing in a test tube rack for 60 minutes with no mixing, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture; (C) the level of NETs generated in whole blood in a serum BCT left standing in a test tube rack for 60 minutes with no mixing, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the baseline level of circulating nucleosomes present in the plasma samples of the same subjects; and (D) the level of NETs generated in whole blood left standing in a test tube rack for 40 minutes with no mixing, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the level of circulating nucleosomes present in serum samples obtained from the same subjects (left for 20 minutes as whole blood with no mixing) and separated at 20 minutes post venepuncture.

The number of diabetic patients measured as having higher levels of NETosis than healthy patients was 2 or less for all the 4 measurements (A) to (D). We note that the method (B) described here is that reported by Sur Chowdhury. However, we did not observe an improved detection of diabetes by this method over the simple measurement of circulating plasma nucleosomes and, in contrast to the reported findings of Sur Chowdhury, we observed no increased NETosis level for RA patients by any of the 4 methods.

The increase of the mean level of nucleosomes or NETs generated for diabetics compared to that generated for healthy subjects was 33%, 25%, 22% and 6% for methods (A), (B), (C) and (D) respectively. This is no higher than the 33% increase observed in simple circulating NETs levels measured in plasma.

Figure 7. The level of NETs generated in whole blood in rotating serum blood collection tubes. The level of NETs generated in the same 17 subjects was measured as: (A) the level of NETs generated in whole blood in a serum BCT left rotating on a tube roller at approximately 60 revolutions per minute (rpm) for 20 minutes, measured as the H3.1 -nucleosome level in serum separated from the whole blood by centrifugation 20 minutes post venepuncture; (B) the level of NETs generated in whole blood left rotating for 60 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture; (C) the level of NETs generated in whole blood left rotating for 60 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the baseline level of circulating nucleosomes present in the plasma samples of the same subjects; and (D) the level of NETs generated in whole blood left rotating for 40 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the level of circulating nucleosomes present in serum samples (also left for 20 minutes as whole blood with rotation) obtained from the same subjects and separated at 20 minutes post venepuncture.

The number of diabetic patients measured as having higher levels of coagulation induced NETosis than any healthy patient was 5, 4, 5 and 7 (out of 10) for methods (A), (B), (C) and (D) respectively. The increase of the mean level of nucleosomes or NETs generated for diabetics compared to that generated for healthy subjects was 57%, 135%, 174% and 370% for methods (A), (B), (C) and (D) respectively. We observed an increased NETosis level for one RA patient by method (A) but not by any of the other 3 methods.

Figure 8. The level of NETs generated in whole blood in shaking serum blood collection tubes. The level of NETs generated in the same 17 subjects was measured as: (A) the level of NETs generated in whole blood in a serum BCT left shaking on an orbital tube shaker at approximately 700 rpm for 20 minutes, measured as the H3.1 -nucleosome level in serum separated from the whole blood by centrifugation 20 minutes post venepuncture; (B) the level of NETs generated in whole blood left shaking for 60 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture; (C) the level of NETs generated in whole blood left shaking for 60 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the baseline level of circulating nucleosomes present in the plasma samples of the same subjects; and (D) the level of NETs generated in whole blood left shaking for 40 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the level of circulating nucleosomes present in serum samples (also left for 20 minutes as whole blood with shaking) obtained from the same subjects and separated at 20 minutes post venepuncture. The results for NETs levels induced in whole blood with shaking for 60 minutes by method (B) were off-scale (higher than the maximum measurable level by the nucleosome immunoassay used) for 4 of the 10 diabetic subjects. The derived methods (C) and (D) were therefore also not measurable, but assumed to be high.

Because quantitative results were not obtained for method (B) for 4 diabetic subjects, these subjects were omitted from calculations for the percentage increase in mean NETS levels presented for Diabetes Mellitus for all methods in Figures 6-8 which was calculated in all cases using the results obtained for the remaining 6 diabetic subjects to ensure comparability.

The number diabetic patients measured as having higher levels of NETosis than any healthy patient was 4 (out of 10) for all the methods (A), (B), (C) and (D). The increase of the mean level of nucleosomes or NETs generated for diabetics compared to that generated for healthy subjects was 86%, 104%, 110% and 114% for methods (A), (B), (C) and (D) respectively. We observed an increased NETosis level for one RA patient by method (A) but not by any of the other 3 methods.

Figure 9. Circulating H3.1 -nucleosome levels in Alzheimer’s Disease patients. EDTA plasma samples collected from 74 subjects including 41 subjects diagnosed with AD and 33 age matched healthy control subjects were analysed for intact cell free nucleosomes containing histone isoform H3.1.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the quantitation of the level or rate of ETs or NETs production or the amount of ETs or NETs produced in a body fluid sample outside of the body by induction of NETosis in the sample as a measurement of the potential of a subject’s body fluid to produce ETs or NETs. Also described herein is a simple accurate method for the quantitation of the potential of a body fluid sample to produce ETs or NETs if induced to do so.

Extracellular traps (ETs) may be formed by a variety of cell types including neutrophils. Therefore, in one embodiment of the methods described herein, the ETs are Neutrophil extracellular traps (NETs). NETs are extracellular traps formed by neutrophils. The terms ETs and NETs are used interchangeably herein. Similarly, the term NETosis as used herein is intended to encompass the process of the extracellular trap production by any cell. Neutrophils occur predominantly in blood and haematopoietic tissues. However, many other body fluids contain neutrophils due to migration of neutrophils to the site of an injury, infection or inflammation. Thus, neutrophils may be found in multiple body fluids including blood, cerebrospinal fluid (CSF), sputum, saliva, urine and stool.

The principle underlying the present invention is to measure the propensity of neutrophils in a body fluid sample taken from a subject to undergo NETosis as an indicator of an increased propensity or risk of the subject to suffer a NETs or NETosis related condition or a NETs or NETosis related complication of a disease or a NETs or NETosis related disease flare or crisis of a disease. Such measurements predict the risk of many disease complications including, without limitation, prediction of the development, or the risk of developing, a dysregulated immune response to an infection (for example leading to a severe COVID-19 infection or sepsis), cancer disease progression, inflammatory or autoimmune disorders, vascular or microvascular disease, thrombosis, microthrombi, disseminated intravascular coagulation (DIC), vasculitis, venous thromboembolism, poor wound healing, diabetic complications including for example vascular complications and diabetic foot complications and many others.

Many drugs affect NETosis. For example, treatment of patients with RA or radiographic axial spondyloarthritis treated with anti-TNF-a drugs (e.g. Infliximab) or anti-interleukin- 6 receptor (anti-l L6R) drugs (e.g. Tocilizumab) targeted at immunological interventions results in reduced NETs levels. Drug treatment of neutrophils isolated from healthy subjects in vitro also results in reduced NETs production (Ruiz-Limon et al, 2020)

The present authors have shown that the propensity of white blood cells to produce ETs in whole blood stimulated by coagulation can be measured accurately to produce reliable results in a simple blood test. We have shown that the range of NETs levels produced ex vivo in whole blood varies in healthy subjects and that the range of NETs levels produced ex vivo in diabetic subjects is elevated. These results are consistent with observations reported for experiments using isolated neutrophils from diabetic subjects cultured in vitro in which NETosis was triggered by calcium ionophore, ionomycin or LPS (Wong et al, 2015).

The level of circulating NETs is reported to be elevated in RA and radiographic axial spondyloarthritis. The level of NETosis that can be induced in neutrophils of these subjects is also elevated. However, treatment of these patients with anti-TNF-a or anti- IL6R drugs results in reduced NETs levels and a reduction in the propensity of neutrophils for NETosis (Ruiz-Limon et al, 2020, Perez-Sanchez et al, 2017 and Sur Chowdhury et al, 2014). In concordance with these findings, the present authors have shown that RA patients treated with such drugs have a normal or elevated level of circulating NETs but a normal or reduced or zero (undetectable) neutrophil propensity for NETosis. Whilst this reduction in neutrophil propensity for NETosis is detected by methods involving isolated neutrophils in vitro, it cannot be detected by other whole blood methods described in the art which therefore are not true measures of neutrophil propensity for NETosis. This is because the level of background or baseline level of circulating NETs already in the blood prior to induction of NETosis ex vivo may vary greatly and is not taken into account. This may be why such methods have not been adopted in clinical practice.

According to a first aspect of the invention there is provided a method for the detection or measurement of the potential of cells in a body fluid, to produce extracellular trap material which comprises comparing the level of extracellular traps (ETs) measured in a first and second sample of the body fluid, wherein the second sample is incubated for a longer period of time than the first sample.

The method of the present invention involves collecting two body fluid samples from a subject. The first sample provides a baseline measure of the level of ETs and/or NETs present in vivo in the subject. NETosis is triggered in a second body fluid sample and the level of ETs and/or NETs present after incubation is measured. The second sample provides a combined measure of the level of the ETs and/or NETs generated ex vivo in the body fluid sample during incubation in addition to the ETs and/or NETs already present at baseline in vivo. The difference between these two measurements provides an accurate measure of the level of NETs generated ex vivo in the body fluid sample during the incubation period and can be used as a measure of the propensity of neutrophils (or other white cells) present in the sample to produce ETs and/or NETs if stimulated to do so by coagulation or drugs. Therefore, in one embodiment, the method comprises comparing the level of ETs measured in the second sample compared to the first sample, in order to detect or measure the potential of cells in the body fluid to produce extracellular trap material.

It will be understood that references herein to “baseline” level, may also include “first”, “initial” or “background” level. The baseline level refers to the level of NETs measures in the first sample, i.e. the sample measure prior to the second sample. Methods of the invention enable the rate of NETosis to be corrected for background by subtraction of the baseline level of circulating nucleosomes present in the samples of the same subjects.

A preferred body fluid sample is a whole blood sample. A first or baseline whole blood sample may be collected in a plasma blood collection tube (BCT) to provide a measure of the level of ETs or NETs in the circulation of a subject prior to initiation of ex vivo NETosis. Alternatively, the first or baseline sample may be collected in a serum BCT which is centrifuged at a preset time following venipuncture (for example at 20 minutes) to provide a measure of the baseline level of ETs or NETs in the sample at a certain time post venipuncture (for example at 20 minutes). It will be appreciated that centrifugation terminates the release of further NETs in the serum sample tested by physically separating the white blood cells from the serum.

The second sample may be collected in a serum sample BCT and incubated (for example for 1 hour) for coagulation triggered NETosis to occur before centrifugation. Using the same example incubation times for illustrative purposes, the difference in levels measured in the first and second samples then represents the ETs and/or NETs formed during a 1 hour incubation (where a plasma first or baseline sample is used) or during a 40 minute incubation (where a serum first or baseline sample is used).

The use of two samples and coagulation triggered NETosis has a number of advantages over methods for neutrophil activation described in the art. The method of the invention is a direct measure of white cell function and does not involve artificial neutrophil stimulation with drugs but is similar to that which occurs naturally in vivo. Similarly, the method of the invention does not involve artificial isolation and culturing of neutrophils but is performed in whole blood which is nearer to the natural environment of white blood cells in vivo.

Methods described in the art for measuring NETosis in whole blood involve only the second part of the method described herein (collection of whole blood in a serum BCT, followed by centrifugation after 1 hour and measurement of NETs in the separated serum) but these methods yield incorrect results, particularly in subjects in whom the in vivo levels are elevated where an incorrectly high result will be measured. Moreover, many or most subjects in whom the propensity for NETosis is likely to be measured will be diagnosed with, or suspected of having, a NETs associated disorder and are therefore likely to have an elevated in vivo level of baseline circulating NETs leading to a false result for NETosis propensity by these methods.

Alternatively, the second sample may be collected in a plasma sample BCT and incubated (for example for 1 hour) for drug triggered NETosis to occur before centrifugation (for example using a PMA, ionomycin, LPS or other NETosis triggering drug). The difference in levels measured in the first and second samples then represents the ETs and/or NETs formed during incubation. It will be understood that references to “trigger” as used herein, includes "induce” or “stimulate".

There are many biomarkers that may be analysed as a measure of NETs. We used immunoassay measurements of nucleosomes containing histone isoform H3.1 (H3.1- nucleosomes) to investigate the kinetic profile of NETosis over 24 hours in whole blood samples collected from healthy subjects in serum BCTs. The results are shown in Figure 1(A) and show that healthy subjects had a varying level or rate of coagulation induced NETosis which continued over the 24 hour period. The increase in H3.1- nucleosome levels was not caused by chromatin release due to cell lysis as no haemolysis was observed. This is surprising because NETosis is described as a process which occurs over 3-4 hours (Kenny et al, 2017). We then performed a similar experiment over 5 days and observed increasing NETosis over 5 days, again with no haemolysis. Therefore, the activity of neutrophils may be measured using measurements of the level of NETs present in a sample at any given time point, without being restricted to analysis at 1 hour post sample collection. This understanding allows flexibility in incubation times and also enables the use of a NETosis inhibitor to terminate NETosis and provide more flexibility to sample collection sites and enable more convenient measurement.

According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of cells in a body fluid, to produce extracellular trap material wherein said method comprises the steps of:

(i) measuring the level of ETs in a first sample of the body fluid;

(ii) incubating a second sample of the body fluid, optionally with addition of a moiety to trigger NETosis;

(iii) measuring the level of ETs in the second sample; and

(iv) using the difference in the measured levels of ETs in the first and second samples as an indicator of the potential of cells in the body fluid to produce extracellular trap material. It will be understood that the first and second body fluid samples described above may be a single sample divided into two parts for the purposes of carrying out the invention as described. Any suitable stimulator of NETosis may be used including PMA, LPS, calcium ionophore A23187 or ionomycin and any such stimulator of NETs may be added to the second sample or to both the first and second samples. NETosis may be terminated prior to measurement of NETs in step (iii), for example by centrifugation of the body fluid sample or by use of a drug molecule or other moiety which inhibits NETosis.

In a preferred embodiment the body fluid sample is a whole blood sample. In this embodiment, NETosis may occur spontaneously (i.e. triggered by coagulation) without the need for a stimulant of NETosis. Therefore, according to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs, wherein said method comprises the steps of:

(i) measuring the level of ETs in the first whole blood sample; or in serum or plasma derived from the first whole blood sample;

In a preferred embodiment, the second whole blood sample is collected in a serum BCT.

In a preferred embodiment, incubation of the second whole blood sample is at least 1 hour in duration.

In a preferred embodiment the second whole blood sample is agitated during the incubation to maintain neutrophils, other white blood cells, platelets and red blood cells in suspension in the sample. It will be understood that references herein to "agitation” include “shaking” or “rotation". In one embodiment, the method comprises vigorous agitation, such as shaking. In an alternative embodiment, the method comprises gentle agitation, such as rotation.

In a preferred embodiment NETosis is terminated prior to measuring the level of ETs and/or NETs, by centrifugation of the whole blood sample and isolation of the supernatant serum. The level of ETs and/or NETs is then measured in the isolated serum.

In one embodiment, the first (i.e. baseline) whole blood sample obtained is collected into a plasma BCT (for example an EDTA or citrate plasma BCT). The whole blood is processed by centrifugation and the isolated plasma is assayed for ETs and/or NETs. Therefore, in one embodiment of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs and/or NETs, wherein said method comprises the steps of:

(i) obtaining a first whole blood sample from the subject in a plasma BCT ;

(ii) optionally adding an inducer of NETosis to said first sample;

(iii) measuring the level of ETs and/or NETs in the first whole blood sample, or in plasma derived from the first whole blood sample;

(iv) obtaining a second whole blood sample from the subject;

(v) optionally adding an inducer of NETosis to said second sample;

(vi) incubating the second whole blood sample to allow NETosis to occur;

(vii) measuring the level of ETs and/or NETs in the second whole blood sample, or in serum or plasma derived from the whole blood sample; and

(viii) using the difference in the measured levels of ETs and/or NETs in steps (iii) and (vii) as a measurement of the potential of the whole blood sample obtained from the subject to produce ETs and/or NETs.

In another embodiment the first whole blood sample is collected into a serum BCT. The whole blood is processed by centrifugation, preferably at a specified time post venipuncture (such as about 20 minutes or about 30 minutes) and the isolated serum is assayed for ETs and/or NETs. Therefore, in one embodiment of the invention there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs, wherein said method comprises the steps of:

(ii) processing the first whole blood sample to terminate NETosis and/or to isolate serum from the sample, and measuring the level of ETs in the serum;

(iv) processing the second whole blood sample to terminate NETosis and/or to isolate serum from the sample and measuring the level of ETs in the serum; and

(v) using the difference in the measured levels of ETs in the first and second samples as a measurement of the potential of the whole blood sample obtained from the subject to produce ETs.

The present authors collected whole blood samples from healthy subjects, subjects with diabetes and subjects with rheumatoid arthritis (RA) in plasma and serum BCTs. The effects of time of incubation with or without mixing or agitating of the whole blood samples in serum BCTs were investigated. Serum BCTs were incubated at room temperature for 20 minutes or for 1 hour by leaving the tubes standing without any mixing, or by rotating the tubes on a standard rocking/rolling roller tube rotator at approximately 60rpm (revolutions per minute) or by shaking the tubes at 700rpm.

The results showed that a longer incubation led to greater production of ETs/NETs. Furthermore, shaking the tubes led to a greater level of NETs/ETs production than rotating the tubes. Overall, longer incubation and agitation led to a greater level of NETs/ETs production than leaving the tubes standing with no mixing.

Therefore, in a further embodiment of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs, wherein said method comprises the steps of:

(i) incubating a first whole blood sample, optionally with agitation of the sample, to allow coagulation to occur, wherein the first whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel; (ii) processing the first whole blood sample to terminate NETosis and/or to isolate serum from the sample, and measuring the level of ETs;

The first baseline sample may also be a serum sample. Therefore, in a further embodiment of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce ETs and/or NETs, wherein said method comprises the steps of:

(i) obtaining a first whole blood sample from the subject in a serum blood collection tube;

(ii) incubating the first whole blood sample, optionally with agitation, shaking or rotation of the sample, to allow coagulation to occur;

(iii) processing the first whole blood sample to isolate the serum and measuring the level of ETs and/or NETs in the serum;

(iv) obtaining a second whole blood sample from the subject in a serum blood collection tube;

(v) incubating the second whole blood sample for a longer time than the first sample, optionally with agitation, shaking or rotation of the sample, to allow NETosis to occur;

(vi) processing the second whole blood sample to isolate the serum and measuring the level of ETs and/or NETs in the serum; and

(vii) using the difference in the measured levels of ETs and/or NETs in steps (iii) and (vi) as a measurement of the potential of the whole blood sample obtained from the subject to produce ETs and/or NETs.

It will be understood that processing a whole blood sample as described herein, refers to a method of separating the cellular and liquid components of whole blood. Any such processing method may be used including centrifugation, gel separation, chromatographic methods or others. This separation physically prevents addition of further ETs or NETs to the liquid (serum or plasma) component. The liquid serum or plasma component also provides a convenient matrix for the measurement of ETs or NETs.

The results of the experiment involving plasma and serum BCT samples taken from healthy, diabetic and RA subjects indicated that the level of H3.1 -nucleosomes measured increased with time of incubation (20m < 60m) and increased with the degree of mixing (no mixing < 60rpm rotation < 700rpm shaking). The H3.1- nucleosome levels for whole blood BCT samples shaken at 700rpm for 60 minutes were too high to be measured by the assay used (off-scale) for 4 of the 10 diabetic subjects tested this way.

The mean level of H3.1 -nucleosomes measured in EDTA plasma samples collected from 5 healthy subjects was 39ng/ml. In samples collected in a serum BCT and incubated 20 minutes post venipuncture before processing by centrifugation, the H3.1- nucleosome level measured was 80ng/ml, 108ng/ml or 183ng/ml when the incubation was performed with no mixing, with rotation at ~60rpm or with shaking at ~700rpm respectively. In samples collected in a serum BCT and incubated 60 minutes post venipuncture before processing by centrifugation, the H3.1 -nucleosome level measured was 113ng/ml, 144ng/ml or 509ng/ml when the incubation was performed with no mixing, with rotation at ~60rpm or with shaking at ~700rpm respectively. Therefore, mixing of whole blood samples during incubation for NETosis increases the level or rate of NETosis that occurs in samples and more vigorous agitation leads to higher levels of NETosis.

This increased measure of nucleosomes was not due to cell lysis. We have observed that even small amounts of cell lysis can lead to observed H3.1 -nucleosomes levels above 1000ng/ml. This indicates that the increase in observed H3.1 -nucleosome levels in serum samples from healthy subjects was not caused by cell lysis in the whole blood prior to processing. In concord with this finding, none of the samples were haemolysed also indicating an absence of cell lysis.

Further, the serum H3.1 -nucleosome levels measured for one of the treated RA patients were consistently low, regardless of rotation or shaking of the serum BCT or the time of incubation. These observations similarly indicate that incubation of whole blood with or without agitation does not result in cell lysis. In order to assess suitable conditions for testing neutrophil activation in a blood test, we investigated sample type, incubation time and the effect of whole blood mixing on measured neutrophil activation in healthy and diabetic subjects. We investigated the number of diabetic subjects determined as having a higher propensity for ex vivo NETs/ETs production than the highest of any healthy subject determined as:

(i) a single timed 20 or 60 minute whole blood incubation assay result,

(ii) the level of NETs/ETs produced in 20 or 60 minutes corrected for baseline determined as the difference in results between a timed whole blood incubation in a serum BCT and a baseline plasma result, or

(iii) the NETs/ETs produced in 40 minutes corrected for baseline determined as the difference between a serum result from a sample centrifuged at 60 minutes post collection and a serum result from a sample centrifuged at 20 minutes post collection.

As a parameter to assess the results, we used the mean observed elevation of neutrophil activation determined for samples obtained from diabetic subjects over that determined for healthy subjects. We also investigated the number of diabetic subjects determined to have a propensity for NETosis that exceeded that of any of the 5 healthy control subjects tested. The results are shown in Table 1.

The EDTA plasma result for ETs/NETs was used as a measure of the in vivo circulating level as a baseline of ETs/NETs present in the samples prior to any ex vivo NETosis. The mean observed circulating NETs/ETs level in plasma was 32% higher in diabetics than in healthy control subjects. Three of ten diabetic patients and one of two RA patients, had ETs/NETs plasma levels that exceeded that of any of the 5 healthy control subjects tested.

The propensity for ETs/NETs production or neutrophil activation level, determined using the method of Sur Chowdhury et al, 2014 identified 2 of 10 diabetic patients with neutrophil activation levels above those of any healthy control subject. The mean increase in measured NETs/ETs production in diabetes was 25%. Therefore, this method provided no improvement in discrimination over a simple plasma measurement of circulating NETs and was not a useful method for measuring neutrophil activation. The experimental condition that generated the highest numbers of diabetics with ETs/NETs generation propensity greater than that of any healthy subject was the level of NETs generated in whole blood left rotating for 40 minutes, measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the level of nucleosomes present in serum samples obtained from the same subjects left as whole blood with rotation and separated at 20 minutes post venepuncture. The results for this method are shown in Figure 7(D) and showed that 7 of 10 diabetic subjects tested were observed to have a propensity to NETosis that exceeded that of any healthy subject. In addition, the mean level of NETs induced in diabetic subjects over healthy subjects was almost 4-fold higher, which was the highest of any of the experimental conditions tested (Table 1).

Table 1. Effect of test conditions on results for neutrophil activation in samples from healthy and diseased subjects

It will be understood that the incubation period used for NETosis to occur in methods of the invention is timed to be the same, or a similar, time period for multiple samples to maintain comparability between samples tested. We have investigated time periods of 20 minutes and 60 minutes. However, the actual time period used for the invention may vary and is not limited to 20 or 60 minutes. In one embodiment, the incubation period is between about 5 minutes and about 24 hours, such as between about 10 minutes and about 2 hours, in particular between about 20 minutes and about 60 minutes. In one embodiment of the invention the incubation period used for NETosis to occur is 1 hour. In further embodiments, the incubation period used for NETosis to occur is shorter than 1 hour, such as approximately 20, 25, 30, 35, 40, 45, 50 or 55 minutes. In further embodiments the incubation period used for NETosis to occur is longer than 1 hour, such as approximately 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes or 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 24 hours or other suitable time period.

In one embodiment, the second sample is incubated for at least 20 minutes ±5 minutes, at least 60 minutes ±10 minutes, at least 3 hours ±15 minutes, at least 4 hours ±20 minutes, at least 6 hours ±30 minutes, at least 12 hours ±1 hour, or at least 24 hours ±2 hours longer than the first sample.

The temperature of incubation may be ambient temperature or the method may be performed at a controlled constant temperature, for example 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C or any suitable temperature. In one embodiment the temperature for incubation is between about 15°C and about 37°C, such as between about 20°C and about 30°C. In a further embodiment the temperature of incubation is 37°C.

This method has the advantage that measurement of the potential or propensity of neutrophils in a subject for NETosis can be determined accurately by taking two serum NETs measurement with two timed incubations with BCT rotation before blood sample processing. This is a simple process that can be easily automated and conducted in hospitals or clinics by phlebotomists, either manually or using a an automated system.

In contrast, the method described by Sur Chowdhury et al., with a 1 hour delay before blood sample processing in serum BCT, does not provide accurate results because there is no correction for a high or low baseline level of circulating nucleosomes in the sample. Coagulation induced ETs release in whole blood may involve, or be influenced by, other cellular, platelet, protein or molecular components of blood. The triggering of the release of ETs and/or N ETs by coagulation is a natural mechanism that also occurs in vivo, so methods of the invention are a good model of the potential or propensity for the production of ETs and/or NETs in vivo in the patient. In contrast, methods for assessing neutrophil activation that involve isolating and culturing neutrophils in vitro are limited to the measurement of NETs produced by neutrophils, and do not measure ETs produced by other cells. Furthermore, the activation of cells and production of NETs is triggered by artificially inducing NETosis using drugs. This is dissimilar to the NETosis process that occurs in vivo and may yield different results than occur for a subject in vivo. In addition, methods involving cell culture and drug stimulation are complex and not suitable for routine clinical use.

The inventors investigated the kinetics of coagulation induced NETosis in whole blood and observed that it continued for at least 24 hours with no sign of slowing (Figure 1A). No hemolysis of the whole blood samples was observed so cells were not disrupted. Therefore, the incubation of samples need not be restricted to 1 hour for NETosis to occur but may be left longer or shorter times.

The incubation time for NETosis may be extended up to 24 hours or beyond for use in the present invention to produce a larger signal and to ameliorate any timing errors. An error of ±5 minutes in 1 hour, as used by Sur Chowdhury et al, is equivalent to a timing error of ±8.3%. This is equivalent to ±20 minutes in 4 hours or ±30 minutes in 6 hours or ±1 hour in 12 hours or ±2 hours in 24 hours. Therefore, the current findings indicate that accurate timing of a 1 hour NETosis incubation may be replaced by simply taking a blood sample at any time and leaving the sample for processing later in the day or on the following day. For example, many blood samples may be collected during a morning for processing during the afternoon or on the following morning.

In one embodiment, the whole blood sample is left for at least about 2 hours following collection. For example, the whole blood sample may be left for at least about 2, such as at least about 2, 3, 4, 5, 6, 8, 12, 24 or 48 hours following collection. In one embodiment, the whole blood sample is left for between 3 and 48 hours, such as between 8 and 24 hours.

We have shown that the level of ETs and/or NETs produced ex vivo by methods of the invention is dramatically increased by agitation of the whole blood. This means that the effect of the baseline level of ETs, NETs, cfDNA or nucleosomes present in the circulation of a subject is minimised (by maximising the ETosis/NETosis signal). Furthermore, the ETosis/NETosis signal can be further increased by extending the incubation time of NETosis used. By using either or both of these means, the effect of the baseline level of ETs, NETs, cfDNA or nucleosomes present in the circulation of a subject can be minimised to the extent that use of a single serum blood tube is facilitated.

Therefore, in a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample to produce ETs and/or NETs, wherein said method comprises the steps of:

(i) incubating the whole blood sample for at least 3 hours with agitation of the sample to enhance the level or rate of ETosis and/or NETosis;

(ii) centrifuging the whole blood sample;

(iii) isolating the serum component of the whole blood sample;

(iv) detecting the level of ETs and/or NETs in the serum; and

(v) using the level of ETs and/or NETs detected as an indicator of the potential of the blood sample to produce ETs and/or NETs.

In one embodiment, step (i) involves leaving the whole blood sample at least 3 hours ±15 minutes or at least 4 hours ±20 minutes or at least 6 hours ±30 minutes or at least 12 hours ±1 hour or at least 24 hours ±2 hours. The chosen length of time may be a sufficient length to allow multiple blood samples to be centrifuged or otherwise processed simultaneously such that the error in the timing of the NETosis/ETosis incubation period is kept within acceptable limits.

In a further aspect of the invention, the potential of a body fluid sample taken from a subject to produce NETs may be quantified by measuring the level or rate of NETosis induced in the sample. The level or rate of NETosis can be measured by commencing coagulation to produce NETs, or by adding a NETosis inducer/stimulant (for example PMA, LPS or ionomycin) and noting the time of induction of ETosis/NETosis. The ongoing ETosis/NETosis may be stopped at any later point in time and the time of stopping is also noted. The time of reaction is calculated as the interval between the two times noted. The level of ETs and/or NETs in the body fluid sample is measured and the level or rate of ETosis and/or NETosis is calculated from the level of ETs and/or NETs measured in the body fluid sample and the time of the ETosis/NETosis reaction. In one embodiment, the body fluid tested may be a whole blood sample with no requirement for a centrifugation step at a pre-determined time. In the case of whole blood, addition of a NETosis activator is optional because ETosis/NETosis can be induced spontaneously by coagulation (for example using a serum blood collection tube). Whole blood is taken into a blood collection tube (for example a serum blood collection tube) and the time of blood draw from the subject is noted. Neutrophils in the sample are induced to produce NETs (either spontaneously by coagulation or by addition of a NETosis activator) and the sample is left for a period of time for the NETs level produced to reach a measurable level. At any later point in time, the whole blood sample is centrifuged and the time of centrifugation is noted. Centrifugation physically separates the serum component of the blood from the cellular component thus preventing the development of further NETs in the serum. The supernatant serum is isolated and assayed for ETs and/or NETs as described herein. The time of reaction is calculated as the interval between the two times noted. The level of NETs in the sample is measured and the level or rate of ETosis and/or NETosis is calculated from the level of ETs and/or NETs produced during the time interval. In the simplest example, a linear level or rate may be calculated as the [level of ETs and/or NETs in the whole blood sample]/[the time of reaction], or more sophisticated non-linear level or rate calculations may be used. The advantage of this method is that the whole blood sample may be centrifuged at any time beyond a minimum time established to be sufficient for the development of a measurable level of ETs/NETs. This may be as little as a few minutes or many hours. The method obviates the need for timing of the centrifugation step. Any sample may be centrifuged at any time point or, for convenience, many or all the samples collected may be centrifuged together at any time point (for example at the end of a working day), so long as the times of incubation are known. The serum transferred following centrifugation may be assayed immediately or may be stored for later assay for cell free nucleosomes, DNA or other NETs or ETs components. Therefore, in a further aspect of the invention, there is provided a method for the detection or measurement of the level or rate of NETosis in a whole blood sample (or the potential of a blood sample to produce NETs), wherein said method comprises the steps of:

(i) inducing NETosis in a whole blood sample and noting the time of induction;

(ii) incubating the sample, optionally with agitation of the sample,

(iii) centrifuging the whole blood sample and noting the time of centrifugation;

(iv) isolating the serum component of the blood sample; (v) detecting or measuring the level of ETs and/or NETs in the serum component;

(vi) determining the time of reaction as the time between the times noted in steps (i) and (iii); and

(vii) using the level of ETs and/or NETs detected, optionally corrected for a baseline level, and the time of reaction as an indicator of the level or rate of ETosis and/or NETosis.

In one embodiment, the whole blood sample is left for at least 1 hour (such as at least 3, 4, 8, 12 or 24 hours) following induction of NETosis for the development of a measurable level of NETs.

In one embodiment, NETosis is induced in the whole blood sample by the coagulation of the blood. In one embodiment coagulation activated NETosis occurs in a serum blood collection tube. In one embodiment, NETosis is triggered by the addition of a NETosis inducing drug to whole blood collected in a plasma BCT. The time of NETosis induction and centrifugation may be noted by starting and stopping a stop-clock or other timer.

It will be understood that the range of levels of ETs and/or NETs produced at any time point by samples taken from healthy or diseased subjects may be determined in advance (for example as shown in Figure 1 for healthy subjects). Therefore, in one embodiment, the level of ETs and/or NETs produced in a test subject during any time period may be compared to the range of levels expected for samples taken from healthy subjects during that same time period to determine if the test level is normal or elevated. Similarly, the level of ETs and/or NETs produced in a test subject during any time period may be compared to the range of levels expected for samples taken from subjects with any particular disease condition during the same time period.

In one embodiment, the method comprises comparing the level of NETs detected in the sample to the level of NETs detected in a reference sample.

In a further aspect of the invention, a comparator reference or calibration sample is used to obviate the need for timing of ETosis/N ETosis incubation. In this aspect neither a fixed incubation time for ETosis/N ETosis, nor the noting of the time of reaction is required. Reference samples used may include low, medium or high reference samples. For example, a low reference sample may be a known healthy blood sample in which the level or rate of NETosis induced by coagulation or other activation, is low. A high reference sample may be a known blood sample in which the level or rate of NETosis induced by coagulation or other activation, is high. ETosis/NETosis calibration samples are samples that exhibit a known level or rate of ETosis/NETosis induction under assay conditions, against which the rate induced in an unknown sample can be calibrated. The calibration or reference sample need not be a body fluid sample but may be a manufactured sample designed to produce ETs and/or NETs when triggered to do so. The test sample and the reference sample(s) are activated at the same time (or near to the same time) to initiate ETosis/NETosis and left for an appropriate time for the development of ETs/NETs. The actual time of incubation for ETs/NETs development is not important. The ETosis/NETosis process is then terminated in the unknown test and reference samples at the same time (or near to the same time). The presence or degree of ETosis/NETosis in the unknown test sample can then be determined in relation to the known ETosis/NETosis level or rate of one or more reference or calibration samples. For example, without limitation, the reference sample may be a sample from a healthy person and a similar signal in the test sample may be used to indicate that the neutrophils of the test subject have normal activity. In contrast, a higher signal in the test sample than the reference sample may indicate that the test subject’s neutrophils are highly pre-activated or pre-disposed to NETosis. Similarly, comparisons may be made using reference samples including pre-activated neutrophils to produce high reference results for comparison to the results of a test subject. The process may also be quantitative. For example, the known level or rate of production of ETs/NETs in a reference sample may be 10ng nucleosomes/hour/ml. If the signal produced by the reference sample in a nucleosome assay in any arbitrary units where NETosis was stopped at any arbitrary time, were 500 and the signal of a test sample were 120, this would indicate that the level or rate of NETosis in the test sample was 2.4ng nucleosomes/hour/ml [=10x120/500], In one embodiment the test sample is a whole blood sample and NETosis is induced by coagulation. In one embodiment NETosis is terminated by centrifugation of the whole blood sample and transferral of the resulting supernatant serum. In one embodiment the reference and test samples are not similar. For example, the test sample may be a blood sample activated by coagulation whilst the reference sample may constitute neutrophils in a cell culture medium activated by addition of PMA, LPS or other NETosis activator. Therefore, in a further aspect of the invention, there is provided a method for the detection or measurement of the level or rate of NETosis, wherein said method comprises the steps of: (i) inducing NETosis in a whole blood sample and in a reference neutrophil sample;

(ii) leaving the blood sample and reference sample for the development of a measurable level of ETs and/or NETs;

(iii) centrifuging the whole blood sample and reference sample;

(iv) isolating the supernatant component of the blood sample and reference sample;

(v) measuring the level of ETs and/or NETs in the supernatant component of the blood sample and reference sample; and

(vi) using the relative levels of ETs and/or NETs measured in the blood sample and the reference sample as an indicator of the level or rate of NETosis that occurred in the blood sample.

The advantages of the method over previous methods described in the art are that isolation and cell culture of neutrophil cells is not required and similarly the precise timing of the centrifugation of blood samples is also not required. These advantages mean that the method of the invention is suitable for use in any clinical setting and amenable to routine clinical blood tests or to routine sample collection by phlebotomists either in hospital or in doctors’ offices.

NETosis in whole blood samples containing the coagulation inhibitor EDTA was also investigated. Whole blood samples were collected into EDTA plasma blood collection tubes and the tubes were left for various times before centrifugation to produce plasma samples. It was observed that EDTA plasma samples have a similar level of NETs as serum centrifuged at 30 minutes and that no spontaneous NETosis occurred in 24 hours in 19 of 20 samples tested (Figure 3). Therefore, in a further aspect of the invention, EDTA (or other inhibitor of coagulation) is added to a whole blood sample that has undergone spontaneous coagulation induced NETosis, as an inhibitor of further NETosis indirectly through inhibition of coagulation. It will be understood that addition of a NETosis inhibitor to a sample to stop NETosis at a certain time point is a simple process that is simple to automate.

Multiple types of body fluid, including without limitation, blood, bronchoalveolar lavage fluid (BALF), sputum, saliva, urine and stool, are known to comprise neutrophils and NETs or ETs. Therefore, in a further aspect of the invention, the potential of a body fluid sample to produce NETs may be quantified by inducing the sample to produce NETs by addition of a NETs inducing agent or other means and leaving the sample to undergo NETosis for a pre-specified time for example at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or more, or any specified time which provides a suitable level of NETs in the sample tested. The optimal time may vary with the sample type and disease tested for.

In one embodiment the NETosis process is terminated by centrifugation. Therefore, in one embodiment there is provided a method for the detection or measurement of the potential of a body fluid obtained from a subject to produce NETs, wherein said method comprises the steps of:

(i) measuring a level (i.e. baseline level) of NETs in a first sample of the body fluid;

(ii) adding an inducer of NETosis to a second sample of the body fluid;

(iv) centrifuging the second sample;

(v) isolating the supernatant component of the second sample;

(vi) measuring the level of NETs in the supernatant of the second sample; and

(vii) using the difference in the levels of NETs measured in steps (i) and (vi) as an indicator of the potential of the body fluid to produce NETs.

In one embodiment the NETosis process is terminated by using an inhibitor of NETosis. Therefore, when the specified time point is reached, the NETosis reaction may be stopped by a chemical method. One chemical method that may be used is to add a NETosis inhibitor drug or moiety. This method is applicable to any body fluid including in whole blood. Therefore, in one embodiment there is provided a method for the detection or measurement of the level or rate of NETosis or the potential of a body fluid sample to produce NETs, wherein said method comprises adding a NETosis inhibitor to the sample following incubation to prevent further NETosis. In one embodiment the body fluid sample is whole blood that was collected in a serum BCT and the NETosis inhibitor is an inhibitor of coagulation that functions by indirect prevention of further NETosis by preventing further coagulation. Any coagulation inhibitor may be used including EDTA, citrate or others.

In a further aspect of the invention, the potential of a whole blood sample taken from a subject to produce NETs may be quantified by taking the whole blood sample into a sample collection tube for stimulation to produce NETs, either spontaneously induced by coagulation or other means, and leaving the whole blood sample to undergo NETosis for a pre-specified time which provides a suitable level of NETs in the sample tested. The optimal time may vary with the disease tested for. When the specified time point is reached, the NETosis reaction is stopped by a chemical method. One chemical method that may be used is to add a NETosis inhibitor drug or moiety which inhibits or prevents NETosis.

In one embodiment the NETosis process is terminated by addition of a NETosis inhibitor. Therefore, in one embodiment there is provided a method for the detection or measurement of the potential of a body fluid obtained from a subject to produce NETs, wherein said method comprises the steps of:

(i) measuring a (baseline) level of NETs in a first sample of the body fluid;

(ii) adding an inducer of NETosis to a second sample of the body fluid;

(iv) adding an inhibitor of NETosis to the second sample;

(v) optionally centrifuging the second sample to remove cellular material or debris;

(vi) measuring the level of NETs in the second sample; and

It will be understood that the first and second samples may be aliquots of a single sample. Therefore, in one embodiment of methods of the invention, the method initially comprises preparing a first and second sample from a body fluid sample obtained from the subject.

INHIBITORS OF NETOSIS

Any moiety that inhibits NETosis in a body fluid sample may be useful in the current invention. Many NETosis inhibitors have been described in the literature and the number is increasing rapidly as the interest in NETosis has increased greatly (partially due to the recent outbreak of COVID-19). Any of these NETosis inhibitors may be used for the invention as described herein. Some examples of NETosis inhibitors described in the literature include, without limitation, anti-citrullinated protein antibodies (Chirivi et al, 2016), GSK484 (an inhibitor of the Protein Arginine Deiminase 4 NETosis pathway), high concentrations of heparin (heparin is reported to be an activator of NETosis at low concentration and an inhibitor of NETosis at high concentrations), anthracycline drugs, including without limitation epirubicin, daunorubicin, doxorubicin and idarubicin, blockers or antibodies to CD32, blockers or antibodies to CD62p and blockers or antibodies to CD162 (Perdomo et al, 2019), indolylmaleimide moieties (Dodo et a/; 2019), leukocyte elastase inhibitor, diisopropyl fluorophosphate, diphenylene iodonium, phenylmethylsulfonyl fluoride, aminoethylbenzene sulfonylfluorid (Farley et a/; 2012) and BMS-P5 (Li et a/; 2020). Sondo et a/; 2019 identified 70 putative inhibitors of NETosis, 22 of which fully inhibited NETosis onset. These included kinase inhibitors, vanilloids (for example, capsaicin and dihydrocapsaicin) and tetrahydroisoquinolines. Similarly, a large number of NETosis inhibitors are disclosed in WO2016/127255. It will be understood that any of these NETosis inhibitors, as well as any yet to be described, may be used in the present invention as described herein.

It will be understood that NETosis is inhibited in a whole blood sample by the addition of an inhibitor of coagulation. Thus, NETosis may be inhibited indirectly by preventing or inhibiting induction of NETosis by coagulation. Many inhibitors of coagulation are known in the art including heparin, citrate and EDTA. Thus EDTA, or other anticoagulant, may be added to a whole blood sample (for example in a serum blood collection tube) after leaving the whole blood sample for NETosis to occur to prevent further coagulation and indirectly inhibit NETosis. Therefore, in one embodiment the inhibitor of NETosis is an anticoagulant.

Moieties such as azide that induce rapid cell death without cell lysis may also prevent further NETosis and be useful for the termination of NETosis in methods of the invention.

Inhibitors of NETosis that fully inhibit or prevent NETosis are particularly useful for preventing NETosis in blood or other body fluid samples for use in the present invention.

SAMPLE MATRIX EFFECTS AND KINETICS OF NETOSIS IN WHOLE BLOOD

The literature is conflicted on the selection of blood sample matrix for use in the measurement of circulating NETs in blood samples (i.e. the actual level of NETs in the circulation in vivo at the time of sample collection). Measurements are described in EDTA plasma, heparin plasma and serum samples (collected with or without gel separation of the cell and serum fractions). However, the preanalytics of blood sample collection have not been reported and there are no standardised sample collection protocols. Many publications state that the measurements were carried out using plasma or serum samples with no information provided on sample preparation presumably because this is not considered relevant (Zuo etal, 2020). Similarly, articles focussed explicitly on the measurement of NETs in biospecimens and which recommend urgent standardization of the methods used as being pivotal to the potential of NETs markers in diagnostics and prognostics, are silent on sample matrix or collection protocol (Rada, 2019, Thalin etal, 2019). Whilst many methods have been reported for research use, none of these methods have been used clinically to aid patients.

Workers in the field have measured NETs, or components of NETs, in a variety of sample matrices including serum collected in (plain) serum tubes or in serum gel separation tubes, EDTA plasma and heparin plasma. The inventors have determined that these matrices are not equivalent and their interchangeable use reflects a lack of understanding of the NETosis process as it occurs post blood collection in the art.

Not only has the kinetics of coagulation-stimulated NETosis in whole blood not previously been considered or investigated, but NETosis has been reported to be prevented by the addition of blood components. Formation by human neutrophils following stimulation with LPS and Cal in cell culture was reported to be prevented by the addition of heat-inactivated fetal calf serum, 0.5% human serum albumin, or 0.5% bovine serum albumin. PMA induced NETosis was not affected so blood components such as human or bovine albumin inhibit NETs formation to different degrees, depending on the NETosis inducer used. In contrast, NETosis of murine neutrophils was inhibited by addition of serum or albumin regardless of the inducer employed (Neubert et al, 2019).

Described herein is an investigation of the kinetics of NETosis stimulated by coagulation in whole blood. Whole blood samples were collected from healthy volunteers in regular serum collection tubes and assayed for the amount of cell free nucleosomes containing histone isoform H3.1 (H3.1 -nucleosomes) as a measurement of the level of NETs production stimulated by coagulation at various time points up to 96 hours. As illustrated by Figure 1 , a similar pattern of continuous NETs production was observed for all 4 subjects but with different NETosis rates observed for different subjects. This indicates that, whilst the propensity of neutrophils for NETosis in the circulation of healthy subjects is low, it none-the-less differs even among healthy subjects. In a separate experiment, the kinetics of NETosis induced by coagulation using serum gel separation collection tubes was also investigated. The result was a slower appearance of the NETs in the serum. Without being bound by theory, the inventors conclude that this was likely caused by the physical barrier separating the serum from the neutrophils which slowed movement of the released NETs into the serum. In addition, it is possible that the physical separation of cells and serum also slows the level or rate of NETs release by preventing contact between neutrophils and some serum components that may be required for, or may accelerate, NETosis. In one embodiment, the sample is collected in a serum separator collection tube comprising a separation gel. In another embodiment, the sample is collected into a container containing an inhibitor of coagulation and a stimulant (or inducer) of NETosis is added to the sample.

The behaviour of plasma EDTA blood collection tubes with respect to the generation of NETs in whole blood before centrifugation was also investigated. It was found that the measured levels of H3.1 -nucleosomes in EDTA plasma did not increase with increasing time of storage of the whole blood sample prior to centrifugation. Therefore, no coagulation induced NETosis occurred during this time in these whole blood samples because coagulation was prevented by the presence of EDTA (Figure 3). This means that EDTA plasma samples taken concurrently to the coagulation induced NETosis test serum samples can be used as a baseline measurement of NETs in the sample prior to stimulation of NETosis by coagulation.

Heparin is known to induce NETosis. Therefore, the level of NETs produced in matched EDTA and heparin plasma samples taken from 5 healthy volunteers at 1 hour post phlebotomy using an assay for H3.1 -nucleosomes for detection of NETs was also investigated and compared. It was found that 2 of the 5 heparin plasma samples produced a high level of NETs, that were not produced in the corresponding EDTA plasma samples taken from the same subjects. One heparin sample showed a low level of NETs production and 2 samples showed little or no NETs production.

The same matched heparin and EDTA plasma samples taken from the 3 volunteers in whom an elevation in NETs was observed in heparin plasma (but not in EDTA plasma) were assayed using two further NETs assays for MPO-DNA and NE-DNA. The results for the 3 samples all showed low levels of NETs in the EDTA plasma samples and elevated levels in the heparin samples as found for H3.1 -nucleosomes, confirming that the chromatin material produced in heparin plasma was NETs. The presence of NETs in the heparin plasma, but not in matched EDTA plasma samples, indicates that the origin of the NETs in the heparin samples was due to NETosis in whole blood after collection of the blood sample. NETosis in heparin plasma blood collection tubes was unlikely to have been caused by coagulation (as coagulation is prevented by heparin) but was directly induced by heparin. Therefore, heparin plasma tubes may be used for methods of the invention. It will be understood that any blood collection receptacle that is compatible with the NETosis process may be used for methods of the invention.

EFFECT OF NETOSIS INHIBITORS

The level of cell free nucleosomes by immunoassay was used as a measure of the clotting induced NETs production in the serum of 4 healthy volunteers with increasing time of clotting up to 24 hours before centrifugation. In the absence of an inhibitor of NETosis, nucleosome levels increased continuously over 24 hours with a level or rate that was variable between different healthy subjects (Figure 1A).

In one aspect of the invention there is provided a reagent or additive for addition to a body fluid sample for the purposes of stopping or terminating NETosis in the sample. In one embodiment the reagent or additive includes a NETosis inhibitor.

ASSAYS FOR NETs

NETs are composed of decondensed or unwound chromatin consisting primarily of strings of nucleosomes with a beads on a string type of structure decorated or granulated with myeloperoxidase (MPO), neutrophil elastase (NE) and other proteins. Each nucleosome consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex are wrapped approximately 145 base pairs (bp) of DNA. Further DNA, that is often referred to as “linker DNA”, connects each nucleosome in a “string” to the next, i.e. the DNA connecting one nucleosome to another in chromosomes. Another histone, H1 , which may be located on the nucleosome outside of the core histones, binds to linker DNA and this may also be present in some nucleosome strings.

There are many assays described in the art for the measurement of NETs. These include without limitation, assays for DNA (particularly cfDNA), histones, nucleosomes (including nucleosomes containing particular epigenetic signals such as citrullinated nucleosomes), MPO or NE assays or nucleosome or DNA adduct assays incorporating MPO or NE. It will be understood that any measure of NETs may be employed in methods of the invention. Therefore, in one embodiment, the level of NETs is measured by detecting the level of cell free nucleosomes and/or cell free nucleosomes containing a particular epigenetic feature, or the level of MPO, NE, cfDNA, MPO-DNA, NE-DNA, MPO-nucleosome adduct or NE-nucleosome adduct. Both cell free MPO- nucleosome adduct and NE-nucleosome adduct assays, utilising one antibody to MPO or NE and another antibody to a histone component of a nucleosome, are novel and not reported in the literature.

In another experiment, serum samples taken from 2 healthy volunteers that were centrifuged 30 minutes and 24 hours after venepuncture, were assayed for NETs using assays for MPO-DNA and NE-DNA. These are both markers specific to NETs derived chromatin. The results were compared to the results of a manual ELISA assay for nucleosomes. The results are shown in Table 3 and demonstrate the same increasing pattern of NETs observed with assays for nucleosomes. This shows that any assay for NETs derived chromatin may be used for methods of the invention.

In a further experiment, the levels of nucleosomes present in plasma taken from animal subjects with elevated levels of circulating NETs were measured and compared to observed nucleosome levels with cfDNA levels measured in the same samples by quantitative PCR. The levels correlated well with a Spearman’s correlation of 96%. Therefore, cfDNA measurements may be used for methods of the invention.

The inventors conclude that the different rates of NETosis observed in different samples are a measure of the potential of the samples to produce NETs if stimulated to do so. In turn this may relate to the propensity of the neutrophils in the samples to undergo NETosis or the “amount or level of pre-activated neutrophils” present in the samples. The meanings of the terms the “potential to produce NETs”, “capacity to produce NETs”, “propensity to produce NETs” and the “amount or level of pre-activated neutrophils”, and the “amount or level of primed neutrophils” in a subject’s blood or sample thereof are therefore equivalent and may be used interchangeably herein. Similarly, the terms “ETs”, “NETs”, “ETs/NETs” or “ETs and/or NETs” may be used interchangeably herein. Similarly, the terms “ETosis”, “NETosis”, “ETosis/NETosis” or “ETosis and/or NETosis” may be used interchangeably herein.

Key advantages to the measurement of the potential of a sample to produce NETs by methods of the invention include method simplicity and ease of automation, the small sample volumes required and the low cost of ELISA methods (as opposed to many of the methods in the literature which utilise fluorescence activated cell sorting methods). Thus, in one aspect of the invention, the method involved may simply be taking whole blood into a serum collection tube, adding the tube to an automated instrument which rotates or shakes the sample for a specific time before centrifuging it. The sample supernatant may then be transferred to an automated immunoassay analyser for analysis. Alternatively, the sample incubation, timed centrifugation and automated immunoassay system may be incorporated into a single instrument in a fully automated manner.

The results herein show that the level or rate of NETosis that can be induced in a sample taken from a subject can be quantified by stimulating the sample to produce NETs and leaving the sample to undergo NETosis for sufficient time to produce a measurable level of NETs. Optionally, further NETosis in the sample may then be prevented by a timed addition of a NETosis inhibitor. Optionally, the sample may then be centrifuged and the serum or plasma removed. The sample may be assayed immediately or stored until assayed for cell free nucleosomes, or other NETs components, as a measure of the level or rate of NETosis induced in the sample.

The baseline or to level of NETs present in the subject may also be measured in a sample taken from the same subject where NETosis is not induced (e.g. EDTA plasma). The level of NETs production measured in a sample may then be corrected for the baseline level of NETs present in the subject by subtracting the baseline level of NETs measured.

In one aspect of the invention, the NETs produced in a time period in a whole blood sample is measured using a baseline at an earlier time. The NETs produced in a sample during 40, 60, 80, 100, 120 minutes or any other time period greater than the baseline time, may be determined in this way.

Measurements of the level of NETs produced in a biological sample may be carried out in principle by measuring any protein or nucleic acid component of NETs. Components measured may include specific NETs component proteins, for example MPO or NE. Similarly, measurements of any chromatin components may be used including without limitation cfDNA, cell free nucleosomes, cell free nucleosomes containing particular histone isoforms or histone post-translational modifications (PTMs), for example citrullinated cell free nucleosomes, cell free MPO-DNA complexes, cell free NE-DNA complexes and many other components. Such assays are well known in the art (Rada, 2019). Histone PTMs and their measurement are described, for example, in W02005/019826. Histone isoforms and their measurement are described, for example, in W02013/030579 and WO2016/067029. Proteinnucleosome adducts and their measurement are described, for example, in WO2013/084002. Many further protein NETs components are known in the art and any may be used for methods of the invention including for example those listed by Bruschi et al, 2019.

NETs may also be measured using chromatin protein binders of nucleosomes, including histone H1 and histone H5.

Mononucleosomes and oligonucleosomes are released into the circulation in vivo by apoptotic or necrotic cells. Holdenrieder & Stieber, 2009 reported that concentrations of nucleosomes in plasma and serum are low in healthy subjects but elevated in various cancers as well as in stroke, trauma, sepsis and autoimmune diseases. This illustrates the need for measurement of the potential of a sample for NETosis as the difference between the level of NETs measured at a particular time point corrected for the baseline level present in the sample. Moreover, whilst the correction required in samples taken from healthy subjects may be low, the findings of Holdenrieder & Stieber indicate that the correction required in samples taken from subjects diagnosed with a wide variety of diseases will be higher leading to error. We have shown in the examples herein that use of baseline corrections is superior to the method described by Sur Chowdhury et al, 2014.

Another method to minimize potential interference from background or baseline levels is by the use of assays utilising markers specific to chromatin of NETs origin (e.g. MPO or NE assays or nucleosome or DNA adduct assays incorporating MPO or NE). Therefore, in further embodiments assays for cell free MPO-DNA, NE-DNA, MPO- nucleosome adduct or NE-nucleosome adduct are used to measure NETs.

In one embodiment, the level of NETs is measured by detecting the level of MPO, NE, cfDNA, cell free nucleosomes and/or cell free nucleosomes containing a particular epigenetic feature.

References to “nucleosome” may refer to “cell free nucleosome” when detected in body fluid samples. It will be appreciated that the term cell free nucleosome throughout this document is intended to include any cell free chromatin fragment that includes one or more nucleosomes.

In one embodiment, the level of NETs is measured by detecting the level of cell free nucleosomes present in the whole blood sample. Methods and uses of the invention may measure the level of (cell free) nucleosomes perse. References to “nucleosomes per se” refers to the total nucleosome level or concentration present in the sample, regardless of any epigenetic features the nucleosomes may or may not include. Detection of the total nucleosome level typically involves detecting a histone protein common to all nucleosomes, such as histone H4. Therefore, nucleosomes perse may be measured by detecting a core histone protein, such as histone H4.

Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous group of chromatin fragments originating from the digestion of chromatin on cell death and include an immense variety of epigenetic structures including particular histone isoforms (or variants), post- translational histone modifications, nucleotides or modified nucleotides, and protein adducts. “Epigenetic features”, “epigenetic signal features” or “epigenetic signal structures” of a cell free nucleosome as referred to herein may comprise, without limitation, one or more histone post-translational modifications, histone isoforms, modified nucleotides and/or proteins bound to a nucleosome in a nucleosome-protein adduct. It will be clear to those skilled in the art that an elevation in nucleosome levels may be associated with elevations in some circulating nucleosome subsets containing particular epigenetic signals including nucleosomes comprising particular histone isoforms (or variants), comprising particular post-translational histone modifications, comprising particular nucleotides or modified nucleotides and comprising particular protein adducts. Assays for these types of chromatin fragments are known in the art (for example, see WO 2005/019826, WO 2013/030579, WO 2013/030578, WO 2013/084002 which are herein incorporated by reference).

In one embodiment, the level of NETs is measured by detecting the level of cell free nucleosomes containing a particular epigenetic feature. In a further embodiment, the epigenetic feature is selected from a histone isoform (such as H3.1), histone post- translational modification (such as citrullination) or protein adduct (such as MPO or NE). In one embodiment, the epigenetic feature of the nucleosome comprises one or more histone variants or isoforms. The epigenetic feature of the cell free nucleosome may be a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform. The term “histone variant” and “histone isoform” may be used interchangeably herein. The structure of the nucleosome can also vary by the inclusion of alternative histone isoforms or variants which are different gene or splice products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be classed into a number of families which are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and publicly available for example in the National Human Genome Research Institute NHGRI Histone Database (Marino-Ramirez et al. The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database Vol.2011. and http://genome.nhgri.nih.gov/histones/complete.shtml), the GenBank (NIH genetic sequence) Database, the EMBL Nucleotide Sequence Database and the DNA Data Bank of Japan (DDBJ). For example, variants of histone H2 include H2A1 , H2A2, mH2A1 , mH2A2, H2AX and H2AZ. In another example, histone isoforms of H3 include H3.1 , H3.2, H3.3 and H3t. In one embodiment, the histone isoform is H3.1. Therefore, methods of the invention may comprise detecting or measuring the level of cell free nucleosomes comprising histone H3.1 in order to determine the level of ETs/NETs.

The structure of nucleosomes can vary by post translational modification (PTM) of histone proteins. PTM of histone proteins typically occurs on the tails of the core histones and common modifications include acetylation, methylation or ubiquitination of lysine residues as well as methylation or citrullination of arginine residues and phosphorylation of serine residues and many others. Many histone modifications are known in the art and the number is increasing as new modifications are identified (Zhao and Garcia, 2015 Cold Spring Harb Perspect Biol, 7: a025064). Therefore, in one embodiment, the epigenetic feature of the cell free nucleosome may be a histone post translational modification (PTM). The histone PTM may be a histone PTM of a core nucleosome, e.g. H3, H2A, H2B or H4, in particular H3, H2A or H2B. In particular, the histone PTM is a histone H3 PTM. Examples of such PTMs are described in WO 2005/019826.

For example, the post translational modification may include acetylation, methylation, which may be mono-, di- or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and/or isomerisation (see Ausio (2001) Biochem Cell Bio 79: 693). In one embodiment, the histone PTM is selected from methylation or citrullination. In a further embodiment, the histone PTM is H3 citrulline (H3cit).

A group or class of related histone post translational modifications (rather than a single modification) may also be detected. A typical example, without limitation, would involve a 2-site immunoassay employing one antibody or other selective binder directed to bind to nucleosomes and one antibody or other selective binder directed to bind the group of histone modifications in question. Examples of such antibodies directed to bind to a group of histone modifications include, for illustrative purposes without limitation, anti-pan-acetylation antibodies (e.g. a Pan-acetyl H4 antibody [H4panAc]), anti-citrullination antibodies or anti-ubiquitin antibodies.

In one embodiment, the epigenetic feature of the nucleosome comprises one or more protein-nucleosome adducts or complexes. A further type of circulating nucleosome subset is nucleosome protein adducts. It has been known for many years that chromatin comprises a large number of non-histone proteins bound to its constituent DNA and/or histones. These chromatin associated proteins are of a wide variety of types and have a variety of functions including transcription factors, transcription enhancement factors, transcription repression factors, histone modifying enzymes, DNA damage repair proteins and many more. These chromatin fragments including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins are described in the art.

In one embodiment, the protein adducted to the nucleosome is selected from: MPO or NE. As described herein, these proteins are associated with NETs and are therefore useful in the measurement of the level of NETs in a sample.

Detecting or measuring the level of the biomarker(s) may be performed using one or more reagents, such as a suitable binding agent. For example, the one or more binding agents may comprise a ligand or binder specific for the desired biomarker, e.g. nucleosomes or component part thereof, an epigenetic feature of a nucleosome, MPO, NE and/or cfDNA.

The detection or measurement may comprise an immunoassay, immunochemical, mass spectrometry, chromatographic, chromatin immunoprecipitation or biosensor method. In one embodiment the detection and/or measurement may comprise an immunoassay such as a homogeneous immunoassay (HIA). In a preferred embodiment, detection and/or measurement may comprise a 2-site immunoassay method for nucleosome moieties. A 2-site immunoassay method for the measurement of nucleosomes may employ any antibodies that bind to any part of a nucleosome or moiety adducted to a nucleosome. Such a method is preferred for the measurement of nucleosomes or nucleosome incorporated epigenetic features in situ employing two anti-nucleosome binding agents or an anti-nucleosome binding agent in combination with an anti-histone modification or anti-histone variant or anti-DNA or anti-adducted protein detection binding agent. Also, detection and/or measurement may comprise a 2-site immunoassay employing a labelled anti-nucleosome detection binding agent in combination with an immobilized anti-histone modification or anti-histone variant or anti-adducted protein binding agent.

The inventors herein used a 2-site immunoassay for detecting H3.1 -nucleosomes by employing an immobilized anti-histone H3.1 antibody directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein to capture clipped and nonclipped nucleosomes, together with a labelled anti-nucleosome antibody directed to bind to an epitope present in intact nucleosomes but not present on isolated (free) histone or DNA nucleosome components. This type of epitope may be referred to as a “conformational nucleosome epitope” herein because it requires the native three- dimensional configuration of the target nucleosome to be intact.

In one embodiment, the method of detection or measurement comprises contacting the body fluid sample with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

In one embodiment, the method of detection or measurement comprises: (a) contacting the sample with a first binding agent which binds to an epigenetic feature of a cell free nucleosome; (b) contacting the sample bound by the first binding agent in step (a) with a second binding agent which binds to cell free nucleosomes; and (c) detecting or quantifying the binding of the second binding agent in the sample.

In another embodiment, the method of detection or measurement comprises: (a) contacting the sample with a first binding agent which binds to cell free nucleosomes; (b) contacting the sample bound by the first binding agent in step (a) with a second binding agent which binds to an epigenetic feature of the cell free nucleosome; and (c) detecting or quantifying the binding of the second binding agent in the sample. In a particular embodiment, the method of detection or measurement comprises: (i) contacting the sample with a first binding agent which binds to an epigenetic feature of a cell free nucleosome, wherein the epigenetic feature is histone H3.1 ; (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent which binds to cell free nucleosomes; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

In one embodiment the immunoassay used for the measurement of nucleosomes or other biomarker is performed using a microfluidic device, for example for near patient use.

In one embodiment the immunoassay used for the measurement of nucleosomes or other biomarker is performed using a lateral flow device, for example for near patient use.

In one embodiment the immunoassay used for the measurement of nucleosomes or other biomarker is a homogeneous immunoassay such as an immunoturbidimetric or immunonephelometric assay.

In further embodiments cfDNA measurements are used to detect ETs and/or NETs. Any suitable cfDNA measurement may be used including, without limitation, intercalating fluorescent methods such as Qubit or PCR or other methods.

Methods of detecting biomarkers are known in the art. The reagents may comprise one or more ligands or binders, for example, naturally occurring or chemically synthesised compounds, capable of specific binding to the desired target. A ligand or binder may comprise a peptide, an antibody or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide, capable of specific binding to the desired target. The antibody can be a monoclonal antibody or a fragment thereof. It will be understood that if an antibody fragment is used then it retains the ability to bind the biomarker so that the biomarker may be detected (in accordance with the present invention). A ligand/binder may be labelled with a detectable marker, such as a luminescent, fluorescent, enzyme or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. Alternatively, ligand binding may be determined using a label-free technology for example that of ForteBio Inc. It will be clear to those skilled in the art that the terms “antibody”, “binder” or “ligand” as used herein are not limiting but are intended to include any binder capable of binding to particular molecules or entities and that any suitable binder can be used in the method of the invention. It will also be clear that the term “nucleosomes” is intended to include mononucleosomes and oligonucleosomes and any protein-DNA chromatin fragments that can be analysed in fluid media. In one embodiment, the binding agent, such as the antibody, specifically binds to the target biomarker. The specificity of an antibody is the ability of the antibody to recognize a particular antigen as a unique molecular entity and distinguish it from another. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.

In one embodiment, the level of NETs is measured by immunoassay, mass spectrometry or a proteomics method. In one embodiment, the level of NETs is measured by a quantification of cfDNA including by quantitative PCR, use of fluorescent intercalating DNA dyes or other methods of DNA quantification.

The immunoassays described herein include any method employing one or more antibodies or other specific binders directed to bind to the biomarkers defined herein. Immunoassays include 2-site immunoassays or immunometric assays employing enzyme detection methods (for example ELISA), fluorescence labelled immunometric assays, time-resolved fluorescence labelled immunometric assays, chemiluminescent immunometric assays, immunonephelometric assays, immunoturbidimetric assays, particulate labelled immunometric assays and immunoradiometric assays as well as single-site immunoassays, reagent limited immunoassays, competitive immunoassay methods including labelled antigen and labelled antibody single antibody immunoassay methods with a variety of label types including radioactive, enzyme, fluorescent, time-resolved fluorescent and particulate labels. All of said immunoassay methods are well known in the art.

For example, detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, LIPLC and other LC or LC MSbased techniques. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used.

STIMULATORS OF NETOSIS

Many chemical activators, stimulants or inducers of NETosis are known in the art and any such stimulant of NETosis may be used for methods of the invention to induce neutrophils or other white cells in body fluids to undergo NETosis. Neutrophils in whole blood may undergo NETosis spontaneously, induced by coagulation, or by addition of a chemical activator of NETosis, either in addition to, or in place of, coagulation. It will be clear to those skilled in the art that stimulants used in whole blood samples in addition to coagulation will lead to serum supernatants after centrifugation and that that stimulants used in place of coagulation will lead to plasma supernatants after centrifugation, for example by addition of a stimulant of NETosis to a whole blood sample collected in an EDTA plasma collection tube (or by adding a NETosis stimulant to a tube prior to adding a whole blood sample to the tube).

Therefore, in a further aspect of the invention, NETosis of neutrophils is stimulated by addition of a substance or moiety that stimulates or induces NETs production in a sample. Such stimulants are known in the art and include, without limitation, any bacterial, fungal or viral pathogen or component thereof (living or dead, active or inactive), or any chemical activator of NETosis including, without limitation, heparin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), reactive oxygen species or moieties that generate reactive oxygen species (for example, hydrogen peroxide or glucose oxidase), or calcium ionophores (Cal).

It will be clear to those skilled in the art that such additives may be used to stimulate NETosis in whole blood samples collected in serum or plasma tubes. This aspect of the invention is useful when collecting a whole blood sample in plasma tubes, or if the investigator wants to increase the level or rate of NETosis in the sample. Therefore, in a further aspect of the invention there is provided a method for the detection or measurement of the potential of a whole blood sample taken from a subject to produce NETs, wherein said method comprises the steps of: (i) obtaining a whole blood sample from the subject;

(ii) adding a stimulant or inducer of NETosis to the sample;

(iii) leaving the sample for sufficient time to generate NETs;

(iv) measuring the level of NETs in the sample; and using the level of NETs measured as an indicator of the potential of the blood sample to produce NETs, optionally including a correction for baseline levels of NETs present in the whole blood sample.

In one embodiment the stimulant of NETosis added is selected from: PMA, LPS, Cal or a bacterial, fungal or viral pathogen or a component thereof. In one embodiment the whole blood sample, to which a stimulant of NETosis is added, is collected in a plasma sample collection tube such as an EDTA, heparin, citrate or other plasma sample collection tube. In some embodiments the stimulant of NETosis added to the whole blood sample stimulates a rapid NETosis.

AUTOMATION

In one aspect of the invention there is provided an automated method for the measurement of the level or rate of NETosis induced in a body fluid sample. In one embodiment there is provided a device (e.g. a robotic device) that terminates NETosis in a sample at a pre-determined time point. It will be understood that such a device may take many forms depending on the embodiment of the invention employed. For example, in one embodiment the device may be a liquid handling or dispensing device that terminates NETosis by the addition of a NETosis inhibitor to the sample. In one embodiment the liquid handing device may initiate NETosis by addition of a NETosis activator. In one embodiment the device may include a NETosis reference sample as described herein. In one embodiment, incubation in the device occurs at constant temperature, for example at 37°C.

In one embodiment, termination of NETosis is performed by centrifugation. In a further embodiment the device may include a centrifuge such that a body fluid sample placed in the device is automatically centrifuged at a pre-determined time point. In one embodiment the body fluid sample is a whole blood sample which is centrifuged by the device at a pre-determined time following venepuncture. In one embodiment, two body fluid samples are automatically centrifuged at two pre-determined time points to measure the difference in NETs levels between them. In one embodiment the device includes a mixing mechanism to maintain blood cells in suspension and accelerate NETosis. In a further embodiment the stimulant of NETosis is added to a whole blood sample to accelerate the NETosis process such that the time required to complete the test is a shorter time than that required for stimulation by coagulation alone.

In a further embodiment there is provided a device which automatically initiates and/or terminates NETosis in one or more samples at one or more pre-determined time points and automatically measures the level of NETs in the sample or samples. The device may be fully automated, for example including liquid handling and/or mixing and/or centrifugation and/or assay and/or result calculation aspects of any method of the invention.

In a further embodiment of the invention, there is provided a device for use in a nonlaboratory setting, for example for home use, bedside use or use in a doctor’s office for detecting or measuring NETosis in a body fluid sample taken from a subject. In one embodiment a finger prick blood sample is added to a home/doctor’s office testing device (for example a lateral flow immunoassay device) for analysis of cell free nucleosomes or other NETs component. The blood sample may be stored prior to adding it to the testing device for analysis to allow NETosis to generate NETs in a time fashion. In one embodiment a stimulant or inducer of NETosis may be added to the blood and the sample left to generate NETs before the blood is added to the testing device. In one embodiment the home/doctor’s office testing device utilises an accelerant of NETosis and/or retards the flow of the blood sample sufficiently to allow for NETosis to occur.

In one embodiment of the invention there is provided a device which automatically terminates NETosis in a sample at a specific time and detects or measures the level of NETs generated in the sample. For example, without limitation, a lateral flow immunoassay device may include a NETosis inhibitor to stop NETosis at a certain position or time point in the device. In another example, a lateral flow device may include a filter to remove red and white blood cells from whole blood at a certain position or time point in the device. The level of NETs may be measured in the device beyond the NETosis inhibitor or beyond the filter, for example by an immunoassay for H3.1 -nucleosomes or other NETs components. The level of NETs measured will represent the level or rate of NETosis that occurred in the sample during the time required for the sample flow to reach the NETosis inhibitor or filter in a lateral flow device. The advantage of this form of automation is low cost ease of use at the point of care.

In a further aspect of the invention there is provided a method or device for the collection and/or analysis of whole blood samples for the measurement of NETosis in a near patient setting (for example at home). Subjects may collect a small whole blood sample using a finger prick device. The method or device may include a filter to remove blood cells and/or an activator of NETosis to initiate NETosis and/or a NETosis inhibitor to stop NETosis at a predetermined point or time. Termination of NETosis may be performed manually or automatically within the device. The device may also include measurement of NETs or the blood sample may be sent away (for example by post) for analysis. In one embodiment a finger prick blood sample is collected and transported to a laboratory in a suitably sized container for analysis. In one embodiment the finger prick blood sample is transported to the laboratory as a blood spot on a solid matrix. In one embodiment the container or spot also contains a preservative or gel. In one embodiment the container or spot also contains a stimulant or inducer of NETosis.

ASSESSMENT AND TREATMENT OF DISEASE STATUS

An elevated potential to produce NETs in cell culture has been shown for neutrophils taken from subjects with a variety of disease conditions. According to a further aspect of the present invention there is provided a method to assess disease conditions by testing the potential of neutrophils present in a blood sample to produce NETs where an elevated potential is associated with and/or causal in a wide variety of disease processes. The disease status of a subject may therefore be ascertained using the potential of the subject’s blood or other body fluid to produce NETs as a biomarker for clinical purposes. Therefore, according to a further aspect of the invention, there is provided a method for the diagnosis, prognosis or monitoring of an actual or suspected disease state or syndrome associated with dysregulated or elevated levels of ETs and/or NETs in subject using a method of the invention.

In a further aspect of the invention, there is provided a method for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) measuring the potential of a body fluid sample obtained from the subject to produce ETs or NETs, according to a method as defined herein; and

(ii) using the potential of the body fluid sample to produce ETs or NETs measured as an indicator of the disease status of the subject. In a further aspect of the invention there is provided a method for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) obtaining a body fluid sample from the subject;

(ii) inducing NETosis in the sample;

(iii) measuring the level or rate of NETosis in the sample by a method described herein; and

(iv) using the level or rate of NETosis measured as an indicator of the disease status of the subject.

In a preferred embodiment the body fluid sample is a whole blood sample and NETosis is induced spontaneously by coagulation. Thus, in a preferred embodiment of the invention there is provided a method for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) obtaining one or more whole blood samples from the subject;

(ii) leaving the whole blood sample(s) to coagulate and generate NETs;

(iii) measuring the level or rate of NETosis or the level of NETs produced in the sample(s) by a method described herein; and

(iv) using the level or rate of NETosis or the level of NETs produced as an indicator of the disease status of the subject.

In one aspect of the invention, there is provided a method for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) measuring the level or rate of NETosis induced, or the level of NETs induced, in a body fluid sample obtained from the subject, according to the method as defined herein; and

(ii) using the level or rate of NETosis or the level of NETs measured as an indicator of the disease status of the subject.

Neutrophils are produced in the bone marrow in huge numbers with a life cycle of less than a day (with a half-life in the circulation of 6-12 hours). In this time, they are released into the blood, migrate to tissues, carry out their functions and are then mostly eliminated by macrophages. During this time neutrophils undergo phenotypic changes leading to a heterogeneous mixture of cell subtypes. The cell subtypes may also be related to age and aged neutrophils may be in an activated state (Rosales, 2018). Neutrophils migrate to many tissues in normal subjects such that they have a dynamic resident neutrophil population. Particularly high levels of neutrophils are found in lung tissue. Moreover, the level of neutrophil cells found in the sputum is elevated in asthma and other lung diseases and different lung diseases are characterised by different neutrophil subtype profiles (Moore et al, 2014). It is well known in the art that viable neutrophil cells are found in small numbers in healthy urine and are visible under the microscope. Urinary neutrophil levels are elevated in a variety of urinary tract diseases involving the kidney or bladder including urinary tract infections, prostatitis, urinary blockages, kidney stones, kidney infections, prostate cancer, bladder cancer, renal cancer and other conditions. Testing for the presence of neutrophils and other leukocytes in the urine is a routine clinical procedure usually performed with a dipstick test for leukocyte esterase. Moreover, urinary neutrophils differ phenotypically to those in the circulation. Thus, any body fluid that contains neutrophils may be used as a sample matrix for methods of the invention.

Therefore, in a further aspect of the invention there is provided a method for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(ii) optionally measuring a baseline level of NETs in a sample;

(iii) optionally, adding a stimulant of NETosis to the sample;

(iv) leaving the sample to generate NETs;

(v) terminating NETosis in the sample;

(vi) measuring the level of NETs in the sample;

(vii) optionally correcting the level of NETs measured in step (vi) for the baseline measured in step (ii); and

(viii) using the level of NETs measured as an indicator of the disease status of the subject.

It will be understood that termination of NETosis may involve any suitable method. For example, without limitation, by separating the cellular component of the sample so that no further NETs may enter the liquid phase of the sample (e.g. by centrifugation), by killing or death of the cellular component (e.g. by poisoning with a chemical moiety such as azide) or by addition of a NETosis inhibitor. In a further aspect of the invention there is provided a method and device for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) placing a whole blood sample from the subject in the device;

(ii) incubating the sample for a pre-determined time to generate NETs;

(iii) terminating NETosis in the sample by means of an automated centrifugation by the device at a pre-determined time;

(iv) isolating the serum component of the blood;

(v) measuring the level of NETs in the serum; and

(vi) using the level of NETs measured as an indicator of the disease status of the subject.

In one embodiment there is provided a method and device for the assessment of a disease status of a subject, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(ii) measuring the level or rate of ETs or NETs production induced in the sample by a method described herein;

(iii) using the induced level or rate of ETs or NETs production measured as an indicator of the disease status of the subject.

The term “identifying” “detecting” or “diagnosing” as used herein encompasses identification, confirmation, and/or characterisation of a disease state. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease. Methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

In one embodiment, the methods described herein may be repeated on multiple occasions. This embodiment provides the advantage of allowing the detection results to be monitored over a time period. Such an arrangement will provide the benefit of monitoring or assessing the efficacy of treatment of a disease state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration, relapse and/or remission. In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the results of the test sample with one or more control(s) and/or with one or more previous test sample results taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the nature or amount of the test results in test samples taken on different occasions.

A change in the result of the test sample relative to the result of a previous test sample taken earlier from the same test subject may be indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder or suspected disorder. Furthermore, once treatment has been completed, the method of the invention may be periodically repeated in order to monitor for the recurrence of a disease.

Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in nonhuman animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances. The methods of the present invention are particularly suited to the assessment of inflammatory or anti-inflammatory effects of therapies and therapeutic substances and for the assessment of the effects of substances on the innate immune system.

In a further embodiment the monitoring of more rapid changes due to fast acting therapies may be conducted at shorter intervals of hours or days.

Detecting and/or quantifying may be compared to a cut-off level. Cut-off values can be predetermined by analysing results from multiple patients and controls, and determining a suitable value for classifying a subject as with or without the disease. For example, for diseases where the level of neutrophil activation is higher in patients suffering from the disease, then if the level detected is higher than the cut-off, the patient is indicated to suffer from the disease. Alternatively, for diseases where the level of neutrophil activation is lower in patients suffering from the disease, then if the level detected is lower than the cut-off, the patient is indicated to suffer from the disease. The advantages of using simple cut-off values include the ease with which clinicians are able to understand the test and the elimination of any need for software or other aids in the interpretation of the test results. Cut-off levels can be determined using methods in the art. Detecting and/or quantifying may also be compared to a control. It will be clear to those skilled in the art that the control subjects may be selected on a variety of basis which may include, for example, subjects known to be free of the disease or may be subjects with a different disease (for example, for the investigation of differential diagnosis). The “control” may comprise a healthy subject and/or a non-diseased subject. Comparison with a control is well known in the field of diagnostics. In one embodiment, the potential of a whole blood sample to produce NETs is elevated compared to the control.

It will be understood that it is not necessary to measure control levels for comparative purposes on every occasion. For example, for healthy/non-diseased controls, once the ‘normal range’ is established it can be used as a benchmark for all subsequent tests. A normal range can be established by obtaining samples from multiple healthy control subjects and testing for the level of neutrophil activation by methods described herein. Results (i.e. neutrophil activation levels) for subjects suspected to have a disease can then be examined to see if they fall within, or outside of, the respective normal range. Use of a ‘normal range’ is standard practice for the detection of disease.

According to a further aspect of the invention, there is provided a method of treating a subject diagnosed with, or suspected of, a disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs using a method of the invention comprising the steps of:

(i) investigating the propensity of a whole blood sample (in particular, white blood cells) obtained from the subject to produce ETs and/or NETs by a method of the invention;

(iii) administering the treatment to the subject.

In a further aspect of the invention there is provided a method for identifying a subject in need of treatment for a disease and administering said treatment, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(iii) using the induced level or rate of ETs or NETs production measured as an indicator that the subject is in need of treatment for a disease; and (iv) delivering the treatment to the subject.

The method of the invention has clinical application in a wide variety of diseases. For illustrative purposes, without limitation, some example applications include:

Diabetes:

In one embodiment, the disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs is diabetes.

Diabetes is associated with impaired wound healing and this leads to diabetic complications such as diabetic foot ulcers and amputations. Wong et al, 2015 reported elevated neutrophil activation levels in cultured neutrophils isolated from human diabetes patients as well as from diabetic mice. Impaired wound healing in diabetic mice is associated with an accumulation of large quantities of NETs in wounds. Moreover, removal of NETs by digestion or impairment of NETs production in PAD4 deficient mice, leads to accelerated wound healing in both normal and diabetic mice, without accumulation of large quantities of NETs in wounds.

We have shown herein that neutrophil activation is elevated in the circulation of human diabetic subjects. We conclude that elevated neutrophil activation levels in diabetics make them susceptible to impaired wound healing through a predisposition to excessive NETs production. It is clear that a functional blood test for the potential of neutrophils to produce NETs has clinical application for the identification and monitoring of subjects at risk of diabetic complications.

According to one aspect of the invention, there is provided use of a cell free nucleosome (in particular, the level of cell free nucleosomes containing histone H3.1) as a biomarker in a body fluid sample (in particular a blood, serum or plasma sample), for the diagnosis or detection of diabetes.

According to a further aspect of the invention, there is provided a method of monitoring or assessing a subject diagnosed with, or suspected of, diabetes using a method of the invention comprising the steps of:

(i) investigating the propensity of a whole blood sample obtained from the subject to produce ETs and/or NETs by a method of the invention; and

(ii) using the results obtained in step (i) to monitor the subject. Monitoring a subject can include, for example, assessment of the onset, progression, stabilisation, amelioration, relapse and/or remission of the disease, monitoring efficacy of a therapy, or monitoring subjects for risk of complications associated with the disease.

Alzheimer’s Disease:

In one embodiment, the disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs is Alzheimer’s Disease.

Systemic inflammation, is involved in, or drives, the initiation and progression of Alzheimer’s Disease (AD). Neuroinflammation and systemic inflammation are connected and systemic inflammation plays a key role in AD pathology that precedes Amyloid- deposition. Moreover, epidemiological evidence indicates that long-term use of anti-inflammatory drugs decreases the risk of developing AD. TN F is a key cytokine involved in NETosis signaling and TNF levels are reported to be elevated in AD and to correlate with disease severity. In addition, the use of anti-TNF therapy leads to a reduction in the incidence of AD (Xie et at, 2022, Ou et al, 2021). Moreover, diabetics and subjects suffering from a variety of inflammatory diseases are at significantly increased risk of developing AD. We note that these subjects also have elevated neutrophil activation levels.

We have shown that NETs levels are elevated in AD and that NETs levels correlate with disease severity. We have also shown that anti-TNF therapy, which reduces AD risk, also dramatically reduces neutrophil activation. We conclude that a blood test for neutrophil activation has application in the identification and risk stratification for people at risk of developing cognitive decline, dementia or AD and for monitoring of cognitive decline, dementia or AD and for monitoring the treatment of cognitive decline, dementia or AD.

Therefore, in one embodiment of the invention the potential of a blood sample to produce NETs may be used as a test to predict, or risk stratify, subjects for their probability of developing a dementia disease and to select subjects for therapy to treat or prevent cognitive decline.

Cognitive decline is an important aspect of frailty. Therefore, in one embodiment of the invention the potential of a blood sample to produce NETs may be used as a test to predict, or risk stratify, subjects for their probability of developing a frailty and to select subjects for therapy to treat or prevent a frailty decline.

Cancer:

In one embodiment, the disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs is cancer.

It is known that NETs are involved in cancer disease progression and in the development of metastatic disease. Blood test measurements of neutrophil activation therefore have clinical application in the monitoring of cancer patients for risk of progression and for identifying subjects in need of treatment to lower their inflammatory status or activation level to prevent or delay disease progression.

Anti-inflammatory therapy monitoring:

NETosis is a key component of the innate immune system and a key component of normal and dysregulated inflammation. The level of neutrophil activation in a subject is therefore a key measure of the immune and inflammatory status of a subject. Therefore, blood test measurements of neutrophil activation have clinical application for monitoring the inflammatory status of a subject and for monitoring the effect of antiinflammatory therapies.

In one embodiment the disease is an autoimmune condition, an inflammatory condition, atherosclerosis, infection, diabetes type I, diabetes type II, cancer, pneumonia, respiratory infections, gout, psoriasis, Systemic Lupus Erythematosus (SLE), rheumatoid arthritis, kidney disease, small vessel vasculitis (SVV), Crohn’s disease, colitis, sickle cell disease, SARS, ARDS, stroke or sepsis. Overproduction of NETs is in many cases involved in complications arising from other disease conditions, for example severe acute respiratory syndrome (SARS), acute respiratory distress syndrome (ARDS) or pneumonia complications of respiratory infections such as influenza or coronavirus. Recently, NETs measurements have been found to be useful in the investigation of patients with COVID-19 infections in whom NETs levels were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air (Delgado-Rizo et al, 2017, Maruchi etal, 2018, Zuo et al, 2020).

NETs play a role in blood coagulation by providing a scaffold for clot formation by platelets, red blood cells, extracellular vesicles, and procoagulant molecules. In addition, NETs enhance coagulation by association with tissue factor and activation the intrinsic coagulation pathway and degrading an inhibitor of the extrinsic coagulation pathway (Zhang et al, 2021).

By promoting coagulation, inappropriate NETs also promote blood vessel occlusion, thrombus formation and propagation of arterial and venous thrombosis and microthrombus formation in microvasculature disease. NETs play a central role in thrombosis and inappropriate or excessive NET formation within the vasculature is causative of the vascular problems and thrombosis and microthrombi that occur in a wide variety of diseases (Thalin et al, 2019). Some examples, without limitation, include the promotion of the vascular problems or thrombosis associated with Type I and Type II diabetes, sepsis, cancer, respiratory infections such as influenza or coronavirus infections, pneumonia, atherosclerosis, coronary thrombi, pulmonary thrombi, stroke and deep vein thrombosis. These vascular problems are in many cases severe, leading to death in many diseases (e.g. in stroke, pneumonia or sepsis) or to amputation in diabetes. A variety of treatment regimes and therapies are available for patients with diseases involving such vascular problems. The central role of NETs in these vascular diseases is further supported by reports that treatment with DNase enzyme to digest NETs is an effective thrombolytic therapy.

A thrombus may be large and may block the flow of blood depriving tissues of normal blood flow and oxygen. If a thrombus moves to another site it is termed an embolism. A thrombus may be diagnosed by compression ultrasonography, computed tomography, magnetic resonance imaging or venography. A blood test for D-dimer, a fibrin degradation product of clotting, can also be used to help in the diagnosis. A thrombus may also be small or a microthrombus. Vascular microthrombotic disease, also called microvascular thrombosis or vascular microthrombosis, is a pathological condition of the microvasculature which affects the capillaries and leads to organ damage of the affected tissues. This may be any tissue and is the origin of the microvasculature problems experienced in diabetes, sepsis, pulmonary microthrombi in pneumonia and respiratory infections, cerebral microthrombi, renal microthrombi etc.

Therefore, in one embodiment of the invention the disease is thrombosis or vascular microthrombotic disease. The method of the invention may be used to detect or monitor subjects suffering from, or at high risk of developing, thrombosis or vascular microthrombotic disease. According to a further aspect of the invention, there is provided a method for the detection or measurement of the potential of a whole blood sample obtained from a subject to produce coagulation induced ETs, wherein said method comprises the steps of:

(i) measuring a baseline level of ETs in a first whole blood sample, or in serum derived from the first whole blood sample, wherein the first whole blood sample has been obtained from the subject in a serum blood collection tube or other suitable vessel;

In one embodiment the disease status identified using a method of the invention is a disease which involves pathologic overproduction of N ETs either locally at a particular organ (as for example occurs in the lungs in pneumonia) or more widely (as may occur for example in sepsis). In one embodiment the NETs may be produced at the site of a cancer and may be involved in promoting the spread of the cancer cells to other locations and/or promoting the establishment of metastatic growth at new locations in the body. Furthermore, thrombosis is a common cause of death among cancer patients.

Therefore, in one embodiment of the invention there is provided a method for identifying a subject suffering from cancer that is at high risk of metastatic spread and/or thrombosis, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(iii) using the induced level or rate of ETs or NETs production measured as an indicator that the subject is at high risk of metastatic spread and/or thrombosis. In a further embodiment of the invention there is provided a method for identifying a subject with a cancer disease who is at high risk of disease progression or relapse, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(iii) using the induced level or rate of ETs or NETs production measured as an indicator of the risk of cancer disease progression or relapse.

In a further embodiment of the invention there is provided a method for identifying a subject in need of treatment for a cancer disease and administering said treatment, wherein said method comprises the steps of:

(i) obtaining one or more body fluid samples from the subject;

(iii) using the induced level or rate of ETs or NETs production measured as an indicator of the need for a cancer treatment of the subject; and

(iv) delivering the treatment to the subject.

References to “subject” , “individual” or “patient” are used interchangeably herein. The subject may be a human or an animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. In some embodiments the invention encompasses animal subjects (wild or domesticated). In some embodiments, the invention relates to veterinary uses including for livestock and companion animals such as cats, dogs, horses, donkeys, rats, rabbits, mice, guinea pigs, sheep, goats, pigs, deer, llamas, cows and cattle. In one embodiment, the subject is a non-human mammal, such as a dog, mouse, rat or horse, in particular a dog.

The methods described herein may be performed in vitro or ex vivo. References to acts carried out on a body fluid sample “obtained” from a subject are intended to encompass acts carried out on a body fluid sample already obtained or “obtainable” from a subject and vice versa.

It will be understood that the embodiments described herein may be applied to all aspects of the invention, i.e. the embodiment described for the uses may equally apply to the claimed methods and so forth. The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES

Example 1

To investigate the kinetics of NETosis in whole blood, we collected whole blood samples from 4 healthy volunteers into serum BCTs (Vacutainer Serum Tubes). The tubes were left at room temperature without mixing for NETosis to proceed for 0.5, 1 , 2, 4 and 24 hours after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring serum supernatant into cryovials which were frozen immediately and stored frozen until assay. The level of NETs measured in serum at each time point represents the level of NETs produced in the whole blood sample by NETosis up to the time at which NETosis was stopped by centrifugation.

Assay measurements for serum nucleosomes containing histone variant H3.1 were performed by immunoassay using an automated immunoassay instrument. Briefly, samples were centrifuged prior to analysis for 2 minutes at 14,000xg to remove any large or particulate material. Calibrant or sample (50pl) was then incubated with an acridinium ester labelled anti-nucleosome antibody (50pl) and assay buffer (1 OOpI) for 1800 seconds at 37°C. Magnetic beads coated with an anti-histone H3.1 antibody (20 l) were added and the mixture was incubated a further 900 seconds. The magnetic beads were then isolated, washed 3 times and magnetic bound acridinium ester was determined by luminescence output over 7000 milliseconds.

The results show that the whole blood H3.1 -nucleosome levels increased continuously over the 24 hour incubation of the samples before centrifugation (Figure 1A). Moreover, the observed level or rate of NETosis varied in the 4 healthy subjects. The level of H3.1 -nucleosomes (Nuc-H3.1) measured for the whole blood samples taken from 4 subjects and incubated 30 minutes or 24 hours prior to centrifugation are shown in Table 2. The level or rate of increase in H3.1 -nucleosomes generated (ng/hour/ml) over the 23.5 hours of the experiment were calculated for each subject as [(level at 24h) - (level at 30min)]/23.5. This can also be expressed in pg/min/ml (Table 2). Table 2. The level of NETs and level or rate of NETosis observed in 4 whole blood samples

The level or rate of NETosis inducible in some of the 4 individuals tested was up to 3.5 times that of others. Thus, even among a healthy cohort there is great variation in the potential of their blood to produce NETs.

As the level or rate of NETosis observed over 24 hours did not appear to be slowing, the experiment was repeated by collecting serum samples from a further 5 healthy volunteers into Vacutainer Serum Tubes. The tubes were similarly left at room temperature without mixing for NETosis to proceed for 0.5, 24, 48, 72 and 96 hours after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring serum supernatant into cryovials which were frozen immediately until assay. The level of NETs was measured in serum at each time point as described above.

The results show that the whole blood H3.1 -nucleosome levels increased continuously over the 4 day incubation of the samples before centrifugation (Figure 1 B). Moreover, the level or rate of NETosis varied in the 5 healthy samples. Therefore, incubation times longer than 1 day are viable for use in methods of the invention. As no hemolysis was observed in any sample taken and the shelf-life of whole blood samples stored at 4°C for blood transfusion is 42 days, we deduce that whole blood samples containing neutrophils may be left for NETosis in methods of the invention for periods of one, or a few days.

As another method to measure kinetics of NETosis in blood samples, bioanalyzer traces for the background cell free chromatin fragments present in samples was taken from 2 healthy volunteers in EDTA plasma (Figure 2A) or in serum where the whole blood was left for NETosis to occur for 4 days prior to centrifugation (Figure 2B). Comparison of the traces shown in Figures 2A and 2B shows that the chromatin fragments produced by NETosis in serum increase over time and comprise a qualitatively different mixture of fragment sizes from those present in plasma.

Example 2

In parallel with the experiment described in Example 1 , we collected further whole blood samples from the same 4 healthy subjects at the same time but with addition of epirubicin to the whole blood in the BCTs immediately following collection to produce a final concentration of 10pg/ml. The tubes were left at room temperature without mixing for NETosis to proceed for 0.5, 1 , 2, 4 and 24 hours after venepuncture (with the tubes described in Example 1). After the allocated time, tubes were centrifuged at 3000xg for 10 minutes and the serum supernatant was transferred into cryovials which were frozen immediately until assay for H3.1 -nucleosomes as described in Example 1.

The results showed that NETosis was prevented by the addition of epirubicin. The range of H3.1 -nucleosome levels measured for the 4 subjects after incubation of whole blood with epirubicin for 30 minutes was 7-13ng/ml (compared to 18-59ng/ml in the absence of epirubicin). After leaving the whole blood to undergo NETosis for 24 hours in the presence of epirubicin the observed H3.1 -nucleosome levels were 7-15ng/ml (compared to 59-162ng/ml in the absence of epirubicin). Thus, addition of epirubicin prevented an increase in the level of H3.1 -nucleosomes over the 24 hour period of the experiment.

Example 3

Whole blood samples were collected into serum BCTs from two healthy volunteers. The tubes were left for 0.5 or 48 hours at room temperature without mixing after venepuncture and then centrifuged at 3000xg for 10 minutes to stop further NETosis. Serum supernatant was transferred into 1 ml Nunc cryovials which were frozen immediately until assay. The serum was assayed for MPO-DNA and NE-DNA by immunoassay as follows. Serum sample (25pl) and buffer (75pl) were added to microtitre plate wells coated with a commercially available antibody directed to bind to MPO or to NE and incubated overnight at 4°C. The serum and buffer were removed and the wells were washed 3 times with a wash buffer. A solution containing an anti- DNA antibody conjugated to horse radish peroxidase was added (1 OOpI) and the wells were incubated at room temperature for 1 hour, following which the anti-DNA antibody solution was removed and the wells were again washed 4 times with a wash buffer. A solution containing 3,3',5,5'-Tetramethylbenzidine (TMB) substrate was added and the wells were incubated for 30 minutes, following which a STOP solution containing 1 M HCI was added to terminate the substrate reaction and the OD of the wells was determined at 450nm. The samples were also tested for H3.1 -nucleosomes using a manual microtiter plate format of the assay for H3.1 -nucleosomes (Nuc-H3.1)

Table 3 shows the OD results obtained for the MPO-DNA, NE-DNA and H3.1- nucleosome assays. The results show that a similar increase in NETs was observed for MPO-DNA, NE-DNA and H3.1 -nucleosomes. This result shows that any NETs component may be measured as a measure of NETs and that any of the NETS assays reported in the literature may be used for methods of the invention. As NE and MPO are NETs specific proteins the results also confirm that the moiety measured by the assay for nucleosomes containing histone isoform H3.1 was all or predominantly related to NETs.

Table 3. Results for 3 different NETs assays in serum with delayed centrifugation.

Example 4

We took whole blood samples from 5 healthy volunteers into serum separator blood collection tubes containing a separation gel which forms a barrier between the serum and the blood cells (Griener Bio-One, Item no: 454214) or into plain BCTs (with no separator gel). The tubes were left for NETosis to proceed for 0.5, 1 , 2, 6 and 24 hours at room temperature without mixing after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring serum supernatant into 1 ml Nunc cryovials which were frozen immediately. Samples were assayed for NETs using a microtiter plate format of the assay for nucleosomes containing histone isoform H3.1 (Nuc-H3.1). As in Example 1 , NETosis was observed for all 5 subjects tested both in plain serum tubes and in serum separator tubes. However, the level or rate of NETosis observed in serum separator tubes was slower than that observed for plain serum tubes (with no separator gel) in every case. We conclude that this slower level or rate of appearance of NETs in the serum is due to the separation of the cells from the serum in these tubes. The amount of NETs produced in serum separator tubes may have been similar to that in serum tubes but the level or rate of movement of NETs from the cell fraction into the serum is slowed by the gel barrier formed between the cells and the serum in the tube.

It will be understood that the slow level or rate of increase in NETs in this experiment will allow the measurement of the potential of a whole blood sample to produce NETs over a longer time period and thereby allowing a wide window for the time of centrifugation, for example a window of ±1 hour rather than ±5 minutes as used by Sur Chowdhury et al, 2014.

Example 5

We collected whole blood samples from 20 healthy volunteers into K2EDTA plasma collection tubes. The tubes were left for NETosis to proceed for 0, 2 or 24 hours at room temperature without mixing after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring plasma supernatant into 1 ml Nunc cryovials which were frozen immediately. Samples were assayed for H3.1 -nucleosomes as described in Example 1. The levels were stable for 19 of the 20 samples and did not increase in whole blood containing EDTA during the 24h period of the experiment (Figure 3). This result confirms that prevention of coagulation using EDTA also prevents NETosis and that the NETosis observed in Example 1 was induced by coagulation. The range of results for healthy subjects was 21-49ng/ml with immediate centrifugation following blood collection (i.e. with no incubation for NETosis).

Example 6

We collected whole blood samples from 9 healthy volunteers into Streck plasma blood collection tubes (STRECK Cell-Free DNA BCT). The tubes were left for NETosis to proceed for 2 or 24 hours at room temperature without mixing after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring plasma supernatant into 1 ml Nunc cryovials which were frozen immediately. Plasma samples were assayed for H3.1 -nucleosomes as described in Example 1. The levels were stable for all samples and did not increase in whole blood during the 24h period of the experiment (Figure 4). This result confirms that prevention of coagulation prevented NETosis and that the NETosis observed in Example 1 was induced by coagulation. The range of results for healthy subjects was 29-40ng/ml.

Example 7

We took whole blood samples from 5 healthy volunteers into heparinized plasma BCTs and into K2EDTA plasma BCTs. The tubes were left for NETosis to proceed for 1 hour at room temperature without mixing after venepuncture. NETosis was stopped by centrifuging the tubes at 3000xg for 10 minutes and then transferring plasma supernatant into 1 ml Nunc cryovials which were frozen immediately. Samples were assayed for H3.1 -nucleosomes using a manual microtiter plate ELISA method.

The levels of NETs produced in the 5 EDTA plasma samples were all low as found in Example 5. The level of NETs produced in the matched heparin plasma samples was unaffected for 2 volunteers (i.e. also low), mildly elevated in one volunteer and highly elevated in 2 volunteers. In these samples, NETosis cannot have been induced by coagulation, so we conclude that it was induced by heparin. Thus, heparin is a stimulator of NETosis and may be used as such for methods of the invention.

Example 8

The matched EDTA plasma and heparin plasma samples taken from the 3 volunteers in whom an elevation in NETs was observed in heparin plasma (but not in EDTA plasma) in Example 7, were re-assayed for MPO-DNA and NE-DNA as described in Example 3. The results for the 3 samples all showed low levels of MPO-DNA and NE- DNA in the EDTA plasma samples and elevated levels in the heparin samples as found for nucleosomes containing histone isoform H3.1 in Example 7.

The results showed that any of the NETS assays reported in the literature may be used for methods of the invention and confirm that the nucleosomes measured by the assay for nucleosomes containing histone isoform H3.1 in heparin samples, in Example 7, were NETs or NETs derived.

Example 9

The levels of H3.1 -nucleosomes present in 47 plasma samples taken from animal subjects with elevated levels of circulating NETs were measured as described in Example 1. The levels of cfDNA were also measured in the same samples by quantitative PCR. The H3.1 -nucleosome and cfDNA levels correlated well linearly with a Spearman’s correlation of 96%. Therefore, cfDNA measurements may be used for methods of the invention.

Example 10

We performed an experiment to investigate the relative merits of a serum or plasma baseline NETs measurement, and also to investigate the effect of gentle or vigorous mixing of the whole blood sample on the level of coagulation induced NETosis during incubation.

Six whole blood samples were collected in serum BCTs from each of 10 healthy volunteers, 10 patients diagnosed with type I or type II diabetes and 2 patients diagnosed with rheumatoid arthritis (RA). An EDTA plasma sample was also collected from each subject. For each subject, the six whole blood samples collected in serum BCTs were treated as follows:

1 . incubated 20 mins standing (without mixing)

2. incubated 60 mins standing (without mixing)

3. incubated 20 mins with rotation at approximately 60 rounds per minute (rpm)

4. incubated 60 mins with rotation at approximately 60rpm

5. incubated 20 mins with shaking at approximately 700rpm

6. incubated 60 mins with shaking at approximately 700rpm

All incubations were performed at room temperature. At the end of the allotted incubation period, the whole blood samples in serum BCTs were centrifuged at 3000xg for 10 mins and the supernatant serum was separated and stored at 4-8°C until assay the following day. The whole blood collected in an EDTA plasma BCT was processed at 20 minutes post venipuncture (along with the 20 minute incubated serum BCT samples) and the separated plasma was stored at 4-8°C until assay the following day for H3.1 -nucleosomes using a manual ELISA assay. The EDTA plasma result for ETs/NETs was used as a measure of the in vivo circulating level as a baseline of ETs/NETs present in the samples prior to any ex vivo NETosis.

The results are shown in Table 1 and Figures 5-8. Briefly, the results showed that the levels of NETosis induced in whole blood samples increased with time, increased with gentle mixing of the blood by rolling at 60rpm during incubation and further increased with more vigorous mixing by shaking at 700rpm. As a parameter to assess the results, we used the mean observed elevation of neutrophil activation determined for samples obtained from diabetic subjects over that determined for healthy subjects. On this measure 6 of 7 highest scoring conditions (of the 18 conditions investigated) were measures that included a baseline correction for the H3.1 -nucleosome level present in the sample as described herein. The method described by Sur Chowdhury scored in the 16th position of the 18 conditions.

We also investigated the number of diabetic subjects determined to have a propensity for NETosis that exceeded that of any of the 5 healthy control subjects tested. On this measure one condition was positive for 7 of 10 diabetic subjects and this was a method that that included a baseline correction for the H3.1 -nucleosome level present in the sample as described herein. Four conditions were positive for 5 of 10 diabetic subjects and 3 of these 4 conditions were methods that that included a baseline correction for the H3.1 -nucleosome level present in the sample as described herein.

The best performing set of conditions, amongst the 18 conditions tested, for measuring the propensity of a blood sample to undergo NETosis produced the results shown in Figure 7(D) where 7 of 10 diabetic subjects tested were observed to have a propensity to NETosis that exceeded that of any healthy subject together with an almost 4-fold increase in the mean level of NETs induced in diabetic subjects over healthy subjects (Table 1). In this method, the level of NETs generated in whole blood left rotating for 40 minutes, was measured as the nucleosome level in serum separated from the whole blood by centrifugation 60 minutes post venepuncture corrected for background by subtraction of the level of circulating nucleosomes present in serum samples (also left for 20 minutes as whole blood with rotation) obtained from the same subjects and separated at 20 minutes post venepuncture.

Sur Chowdhury reported that neutrophils from RA cases exhibited increased spontaneous NET formation in whole blood (over 60 minutes without baseline correction). We also measured the propensity for NETosis of whole blood samples taken from 2 subjects diagnosed with RA. In contrast to the findings of Sur Chowdhury, we observed no elevation in the propensity for NETosis in the 2 RA cases we investigated. Surprisingly, one of the two RA cases we investigated appeared to have a near zero propensity for NETosis. However, further investigation revealed that, whereas the cases investigated by Sur Chowdhury had active RA disease without current medication, the 2 RA cases investigated here were volunteers in remission. Moreover, the RA case in whom the NETs production induced by coagulation in whole blood was near zero, was under current anti-TNF-a treatment which is known to inhibit NETosis. This finding illustrates clearly that methods of the invention may be used to monitor the disease state of patients with inflammatory and/or autoimmune conditions and to test or monitor the efficacy of anti-inflammatory treatments.

The samples collected in this Example 10 were also tested for cfDNA content to confirm the neutrophil activation results using a different and independent measurement parameter for NETs (results not yet available at the time of writing).

Example 11

Whole blood samples are collected serially at 3 month intervals from patients diagnosed as suffering from diabetes. The patients are monitored over time for an increase in the potential of their blood to produce NETs, measured as described in Example 10 above, and the results are correlated to diabetes disease progression to the development of diabetic complications including the development of vascular or microvascular disease, diabetic foot ulcers and cases that progressed to a requirement for amputation. Diabetic control is ascertained by glycated hemoglobin (HbA1c) levels as well as glucose levels and clinical parameters.

The results show that elevation of the potential of blood samples to produce NETs is prognostic and precedes, and is predictive of, disease progression to the development of diabetic complications including the development of vascular or microvascular disease, diabetic foot ulcers and cases that progress to a requirement for amputation.

The results also show an association between the elevation of potential of blood samples to produce NETs with diabetic control. Therefore, neutrophil activation measurements may be used to risk stratify diabetic patients and to ascertain both the degree to which the disease control is managed as well as the outcome of good or poor control on the immune status of the subject.

Example 12

EDTA plasma samples were collected from 74 subjects including a cohort of 41 subjects diagnosed with AD and 33 age matched healthy control subjects. Of the 41 subjects with an AD diagnosis, 16 were diagnosed with mild AD, 12 with moderate AD and 13 with severe AD. The plasma samples were analysed for intact cell free nucleosomes containing histone isoform H3.1 using an automated chemiluminescence immunoassay employing an anti-histone H3.1 antibody coated to magnetic particles in combination with a chemiluminescent labelled anti-nucleosome antibody. The results are shown in Figure 9.

It is known that diabetic patients are at increased risk of cognitive impairment and dementia or Alzheimer’s Disease (AD) (Xue et al, 2019). We observed that NETs levels, as measured by H3.1 nucleosome levels, were elevated in samples taken from patients diagnosed with AD (Figure 9). Moreover, the degree of elevation correlated with disease severity. The authors reasoned that the disease stage dependent elevation in circulating NETs in AD is associated with an increased propensity for NETs production in these subjects which also relates to the increased risk of dementia and AD among diabetic patients in whom we have observed increased levels of NETs and neutrophil activation.

Therefore, neutrophil activation measurements methods of the invention may be used to risk stratify subjects for risk of developing dementia or AD and to monitor disease progression and treatment.

Whole blood samples are collected serially annually from patients suspected of suffering, or diagnosed as suffering from Alzheimer’s Disease (AD). The patients are monitored over time for an increase in the potential of their blood to produce NETs, measured as described in Example 10 above, and the results are correlated to AD disease progression from no disease to mild to moderate to severe AD.

The results show that the potential of blood samples to produce NETs is elevated in AD and that the elevation is disease stage dependent. An elevated potential of blood samples to produce NETs is prognostic and precedes, and is predictive of, AD disease progression and/or cognitive decline.

Example 13

It is well known that the mortality rate of COVID-19 infections is much higher among diabetic patients than among subjects without diabetes who contract the infection (Wu et al, 2021). This is one example of a more general finding that diabetic patients who contract an infection are more likely to develop and die from immune related complications involving dysregulation of the innate immune system and dysregulated excessive NETs release, particularly from pneumonia or sepsis (Bertoni et al, 2001). The authors reasoned that the observed 370% increase in the propensity of neutrophils to produce NETs in diabetes patients is causative of this increased risk of developing dysregulated immune complications involving dysregulated excessive NETs release, multiple organ damage or failure and death. Therefore, the methods of the invention may be used prognostically to predict or risk stratify a subject for the risk that an infection, if contracted, may lead to dysregulated excessive NETs release, sepsis, pneumonia, ARDS, SARS, thrombosis, vascular disease and/or other NETs related conditions.

Whole blood samples are collected serially at daily intervals from patients suspected as at risk of developing sepsis. The patients are monitored over time for an increase in the potential of their blood to produce NETs, measured as described in Example 10 above, and the results are correlated to disease progression to the development of symptoms characteristic of the sepsis syndrome (for example the clinical parameters that comprise the SOFA or APACHE score).

The results show that elevation of the potential of blood samples to produce NETs is prognostic and precedes, and is predictive of, disease progression to the development of increasing severity of sepsis syndrome or disease. Therefore, in one embodiment of the invention the potential of a blood sample to produce NETs may be used as a test to predict, or risk stratify, subjects diagnosed with cancer for their probability of suffering disease progression.

Example 14

The literature reports an association of NETs with cancer and cancer progression.

Subjects diagnosed with diabetes are at increased risk for cancer. The highest risks are for liver, pancreatic, colorectal and endometrial cancer. The authors reasoned that the increased risk of cancer among diabetic patients relates to the elevated potential of neutrophils to produce NETs in diabetic subjects.

Whole blood samples are collected serially at 3 month intervals from patients diagnosed as suffering from a primary stage I, stage II, or stage III cancer disease. The patients are monitored over time for an increase in the potential of their blood to produce NETs, measured as described in Example 10 above, and the results are correlated to cancer disease progression to stage IV metastatic disease.

The results show that elevation of the potential of blood samples to produce NETs is prognostic and precedes, and is predictive of, disease progression to stage IV metastatic cancer. REFERENCES

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Claims

1 . A method for the detection or measurement of the potential of cells in a body fluid to produce extracellular trap material which comprises comparing the level of extracellular traps (ETs) measured in a first and second sample of the body fluid, wherein the second sample is incubated for NETosis to proceed for a longer period of time than the first sample.

2. The method according to claim 1 , wherein the difference in the level of ETs measured in the first and second sample is the measurement of the potential of the sample to produce ETs.

3. The method according to claim 1 or claim 2, wherein the body fluid is whole blood.

4. The method according to any one of claims 1 to 3, wherein the ETs are neutrophil extracellular traps (NETs).

5. The method according to any one of claims 1 to 4, wherein NETosis is triggered by coagulation.

6. The method according to any one of claim 1 to 4, wherein the second sample is incubated in the presence of a moiety to trigger NETosis.

7. The method according to claim 6, wherein the moiety to trigger NETosis comprises phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), reactive oxygen species or calcium ionophores.

8. The method according to any one or claims 1 to 7, wherein the first and/or second sample is agitated.

9. The method according to any one of claims 1 to 8, wherein a NETosis inhibitor is added to the first and/or second sample prior to detecting the level of ETs.

10. The method according to any one of claims 1 to 9, wherein the first and/or second sample is centrifuged and the supernatant is collected prior to detecting the level of ETs.

11 . The method according to any one of claims 1 to 10, wherein the second sample is incubated for at least 20 minutes ±5 minutes, at least 60 minutes ±10 minutes, at least 3 hours ±15 minutes, at least 4 hours ±20 minutes, at least 6 hours ±30 minutes, at least 12 hours ±1 hour, or at least 24 hours ±2 hours longer than the first sample.

12. The method according to any one of claims 1 to 11 , wherein the body fluid is whole blood and the sample is collected in a serum blood collection tube (BCT), such as a serum separator collection tube comprising a separation gel.

13. The method according to any one of claims 1 to 12, wherein the sample is collected into a container containing an inhibitor of coagulation and a stimulant of NETosis is added to the sample.

14. The method according to any one of claims 1 to 12, wherein the level of ETs is measured by detecting the level of myeloperoxidase (MPO), neutrophil elastase (NE), cell free DNA (cfDNA), cell free nucleosomes and/or cell free nucleosomes containing a particular epigenetic feature.

15. The method according to claim 14, wherein the level of ETs is measured by detecting the level of cell free nucleosomes present in the sample.

16. The method according to claim 15, wherein the epigenetic feature is selected from a histone isoform (such as H3.1), histone post-translational modification (such as citrullination) or protein adduct (such as MPO or NE).

17. The method according to any one of claims 1 to 16, wherein the level of ETs is measured by immunoassay, mass spectrometry or a proteomics method.

18. The method according to any one of claims 1 to 17, wherein the level of ETs in the sample is detected using an immunoassay for cell free nucleosomes containing histone isoform H3.1.

19. The method according to any one of claims 1 to 16, wherein the level of ETs in the sample is detected using an immunoassay for cell free nucleosomes containing histone isoform H3.1.

20. A method for the diagnosis, prognosis or monitoring of an actual or suspected disease state or syndrome associated with dysregulated or elevated levels of ETs and/or NETs in subject using a method according to any one of claims 1 to 19.

21. A method for assessing the anti-inflammatory effect of a therapy in a subject comprising the steps of:

(i) administering the therapy to the subject;

(ii) investigating the propensity of a body fluid sample obtained from the subject to produce ETs and/or NETs using a method according to any one of claims 1 to 19, to assess the inflammatory status of the subject on one or more occasions;

22. A method of treating a subject diagnosed with, or suspected of, a disease condition or syndrome associated with dysregulated or elevated levels of ETs and/or NETs, comprising the steps of:

(i) investigating the propensity of a whole blood sample obtained from the subject to produce ETs and/or NETs by a method according to any one of claims 1 to 19;

(iii) administering the treatment to the subject.

23. The method according to claim 22, wherein the disease condition is diabetes, dementia or cancer.