Method using nucleosome levels

Abstract

The present invention relates to method of managing clinical pathways for a patient admitted to hospital, comprising measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and determining the health care actions for the patient based on the level of nucleosomes measured in the sample.

Description

[0001] VOL-C-P3782PCT

[0002] METHOD USING NUCLEOSOME LEVELS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to measuring levels of cell free nucleosomes, including NETs, to assist in managing the clinical pathway for patients, particularly upon admission to hospital.

[0005] BACKGROUND OF THE INVENTION

[0006] Sepsis remains a leading cause of morbidity and mortality in critically ill patients, with an estimated 49 million cases and 11 million deaths worldwide annually (Rudd et al. (2020) The Lancet 395:200-211). Early recognition and timely intervention are crucial for improving outcomes, yet the heterogeneous nature of sepsis makes early diagnosis challenging (Prescott and Angus (2018) JAMA 319:62-75). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) emphasise organ dysfunction as a critical feature. However, identifying the transition from infection to sepsis and predicting progression to septic shock and multiple organ failure remain significant clinical challenges (Singer et al. (2016) JAMA 315:801). Acute kidney injury (AKI) is a common and serious complication in patients with sepsis, associated with increased mortality, longer intensive care unit (ICU) stays, and greater healthcare costs (Uchino et al. (2005) JAMA 294:813-818; Chertow et al. (2005) J Am Soc Nephrol JASN 16:3365-3370; and Monard et al. (2023) BMC Nephrol 24:343). The incidence of AKI in septic patients is high, with studies reporting rates between 40-70% (Uchino et al. (2005) supra, Bellomo et al. (2012) Lancet Lond Engl 380:756-766).

[0007] Recent advances in understanding sepsis pathophysiology have highlighted the role of the dysregulated host immune response, including the formation of neutrophil extracellular traps (NETs) (Papayannopoulos (2018) Nat Rev Immunol 18:134-147). NETs, composed of extruded nuclear chromatin decorated with histones and granular proteins, serve to trap and neutralise pathogens but can also contribute to organ damage when excessively produced or inadequately cleared (Czaikoski et al. (2016) PloS One 11 :e0148142). Histones, particularly histone 3 (H3), have also been implicated as critical mediators of endothelial damage and organ dysfunction in sepsis (Abrams et al. (2019) Am J Respir Crit Care Med 200:869-880). Nucleosomes, the fundamental units of chromatin consisting of DNA wrapped around histone octamers, are released during NET formation and cell death (Marsman et al. (2016) Cell Death Dis 7:e2518-e2518). The H3.1 variant of histone H3 is particularly abundant in mature neutrophils. The utility of H3.1 nucleosomes in identifying early sepsis and predicting disease progression has not been thoroughly investigated. VOL-C-P3782PCT

[0008] There remains a need in the art to provide simple, rapid, cost-effective methods to identify and prioritize individuals upon admission to hospital so that the health care actions can be assigned quickly and efficiently.

[0009] SUMMARY OF THE INVENTION

[0010] According to one aspect of the invention there is provided a method of managing the clinical pathway for a patient admitted to hospital, comprising:

[0011] (i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and

[0012] (ii) determining the health care actions for the patient based on the level of nucleosomes measured in the sample.

[0013] According to another aspect of the present invention there is provided a method of determining the clinical pathway for a patient admitted to hospital, comprising:

[0014] (i) providing a body fluid sample obtained from the patient upon admission to hospital;

[0015] (ii) contacting the body fluid sample with an antibody that specifically binds histone H3.1 ; and

[0016] (iii) measuring the level of nucleosomes present in the body fluid sample by detecting the level of binding of the antibody that specifically binds histone H3.1 ; wherein the level of nucleosomes measured in the body fluid sample is used to determine one or more health care actions that form the clinical pathway for the patient.

[0017] According to another aspect of the present invention there is provided a method of predicting a risk of mortality for a patient admitted to hospital, comprising:

[0018] (i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and

[0019] (ii) determining the risk of mortality of the patient based on the level of nucleosomes measured in the sample.

[0020] According to another aspect of the present invention there is provided a method of determining the likelihood of a patient requiring renal replacement therapy (RRT) comprising measuring the level of neutrophil extracellular traps (NETs) in a body fluid sample obtained from the patient.

[0021] According to another aspect of the present invention there is provided the use of a level of NETs in a body fluid sample as a biomarker, to identify patients with a high likelihood of requiring RRT within 28 days of admission to hospital. VOL-C-P3782PCT

[0022] According to another aspect of the present invention there is provided a kit for detecting a panel of biomarkers in a body fluid sample, comprising reagents suitable for determining levels of the panel of biomarkers, wherein the biomarkers comprise NETs, urine output and platelets; optionally comprising instructions for use of the kit in the method defined herein.

[0023] BRIEF DESCRIPTION OF FIGURES

[0024] Figure 1. Kaplan-Meier plots of: (A) overall survival; (B) 28-day survival and (C) 28-day survival after dividing the population equally into quintiles, for patients according to H3.1- nucleosome levels at admission.

[0025] Figure 2. Graph plotting initial H3.1 -nucleosome levels against days to death. Initial H3.1- nucleosome readings above 10,000 predict mortality within 14 days. Overall survivors were not plotted.

[0026] Figure 3. Analysis of 28-day survival model based on maximum serum lactate (12.9 mmol / L), urea (16 mmol / L), 24 h urine (1 ,035.8 mL), platelet count (102 x 109 / L) and H3.1 -nucleosome levels (6,800 ng / mL). (A) Box and whisker plot, and (B) Receiver operating characteristic (ROC) curve.

[0027] Figure 4. AUC-ROC analysis of prediction of renal replacement therapy (RRT) for (A) initial H3.1 -nucleosome levels, (B) initial platelet counts and (C) 24 h urine.

[0028] Figure 5. Scatterplot by diagnosis of (A) initial H3.1 -nucleosome levels and platelet counts and (B) initial H3.1 -nucleosome levels and 24 h urine.

[0029] Figure 6. Ability of 24 h urine, initial H3.1 -nucleosome levels and platelet counts to predict the need for RRT. (A) Results shown as box representing 25th-75th percentile with median and individual values for patients. (B) ROC curve analysis, with 95% Cl, of the clinical model using initial H3.1 -nucleosome levels, platelet count and 24 h urine in combination to determine time to RRT.

[0030] Figure 7. Kaplan-Meier analysis of the ability of the clinical model to determine time to RRT. As indicated by the key, the top line is the “Score 0” group, the second line down is the “Score 1” group, the third line down is the “Score 2” group and the bottom line is the “Score 3” group. VOL-C-P3782PCT

[0031] Figure 8. Kaplan-Meier analysis of the ability of the clinical model to determine time to RRT with initial H3.1 -nucleosome levels removed from the model. As indicated by the key, the top line is the “Score 0” group, the middle line is the “Score 1” group, the bottom line is the “Score 2” group.

[0032] DETAILED DESCRIPTION

[0033] The present invention utilises elevated levels of cell free nucleosomes, including NETs, to assist in managing the clinical pathway for patients, particularly upon admission to hospital.

[0034] Nucleosomes are released into the circulation on fragmentation of chromatin on cell death. Many infections, such as viral infections, initiate cell death through a variety of mechanisms (cell binding and entry, endosomal TLR3 activation and gene expression) thereby increasing the number of circulating nucleosomes in the blood (Danthi et al., Annu. Rev. Virol. (2016) 3: 533-53). In NETosis, post-translational histone modifications, such as acetylation or hypercitrullination of histones H3 and H4 (Wang Y et al., J. Cell Biol. (2009) 184(2): 205-213), promote decondensation of chromatin which is released into circulation together as a first line response to infection. However, extracellular nucleosomes and neutrophil extracellular traps (NETs) can cause severe complications if not cleared rapidly. For example, nucleosome binding to the glomerular membrane is associated with kidney damage in lupus (Kalaaji et al., Kidney Int. (2007) 71(7): 665-672), whilst NETs have been shown to intensify pulmonary injury during viral pneumonia (Ashar et al., Am. J. Pathol. (2018) 188(1): 135-148). Indeed, host directed NET toxicity is associated with respiratory distress, occlusion of narrow airways, endothelial and epithelial cell damage, inflammatory response and thrombus formation and other pathologies (Marcos et al., Nat. Med. (2010) 16: 1018-23; Hoeksema et al., Future Microbiol. (2016) 11 : 441-53).

[0035] The present inventors have identified that nucleosome levels detected in patients upon admission to hospital could provide critical information to help manage and prioritise appropriate treatment steps to provide a suitable clinical pathway.

[0036] Therefore, according to one aspect, there is provided a method of managing the clinical pathway for a patient admitted to hospital, comprising:

[0037] (i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and

[0038] (ii) determining the health care actions for the patient based on the level of nucleosomes measured in the sample. VOL-C-P3782PCT

[0039] Hospitals contain different departments where patients are housed depending on the nature and severity of the disease or illness. In a preferred embodiment, the patient is admitted to an intensive care unit (ICU) of the hospital. ICUs are specialist hospital wards that provide treatment and monitoring for patients who are seriously ill, particularly those that require intensive treatment and close monitoring. An ICU may also be referred to as a critical care unit (CCU) or intensive therapy unit (ITU).

[0040] Methods of the invention are used to establish the treatment steps (e.g. health care actions) for a patient upon admission to hospital. Reference herein to “clinical pathway” (which may also be referred to as a “care pathway”) refers to the clinical guidelines of diagnosis or treatment assigned to a patient, and includes, for example, an order of diagnosis and treatment and a progression order of a disease. It details the steps in a course of treatment or care in a plan, pathway, algorithm, guideline, protocol or other “inventory of actions” (i.e. the intervention has time-frames or criteria-based progression). It aims to facilitate communication among the healthcare team and with the patient and their family. It also enables coordination of the care process by providing an outline of treatment and sequence of activities, for use by a potentially multidisciplinary care team.

[0041] Reference herein to “health care actions”, which may also be referred to as “treatment steps”, refers to the actions taken by the healthcare team to treat, monitor and / or manage a patient after they are admitted to hospital.

[0042] In one embodiment, the health care actions comprise one or more ICU medical interventions. Medical interventions (or “therapeutic measures”) are selected based on the clinical pathway established for the patient and may include: administration of one or more treatments such as intravenous fluids, vasopressors (e.g. norepinephrine), corticosteroids (e.g. hydrocortisone) and / or antimicrobial agents; fluid resuscitation; renal replacement therapy, such as dialysis; and / or invasive / mechanical ventilation. In a further embodiment, the ICU medical intervention is selected from renal replacement therapy (RRT), invasive ventilation and / or vasopressors.

[0043] In one embodiment, the patient is suffering from, or is likely to be suffering from, sepsis or septic shock.

[0044] Sepsis is a severe inflammatory medical condition that can lead to hemodynamic shock and acute organ failure and is a leading cause of hospital mortality. Sepsis can involve any or all of low blood pressure, accelerated heart rate, pain, fever with sweaty skin and feeling cold, shortness of breath and disorientation or confusion. The condition of sepsis patients may VOL-C-P3782PCT deteriorate rapidly over hours into septic shock with low blood pressure, stroke, respiratory failure, heart failure, or multiple organ failure. Sepsis requires immediate treatment with intravenous fluids and antimicrobials often in an intensive care setting. Mechanical ventilation and dialysis may be needed to support the function of the lungs and kidneys, as well as preventive measures for thrombosis. Patient outcome depends on prompt diagnosis and early treatment. However, sepsis is not easy to diagnose, especially in critically ill patients.

[0045] In sepsis, an inflammatory stimulus triggers a severe inflammatory response characterised by a cytokine storm with elevated levels of circulating cytokines as well as elevated production of NETs and elevated levels of circulating cf-nucleosomes and cfDNA. Elevated cytokine and NETs production by NETosis is pathological and has been found to lead to thrombosis, low blood pressure, high blood lactate and low urine output, leading eventually to respiratory distress, loss of consciousness and multiple organ failure.

[0046] Sepsis may arise from non-infectious inflammatory stimuli such as polytrauma, surgery, pancreatitis or burns. More commonly, sepsis may be caused by infectious inflammatory stimuli such as bacterial, fungal, viral or protozoan infection. Influenza and COVID-19 are examples of well-known viral infections that can lead to systemic inflammatory response syndrome (SIRS). Sepsis progresses from SIRS, to sepsis, severe sepsis and then septic shock.

[0047] Rapid early identification of these patients to enable timely treatment is important because their condition may deteriorate rapidly and failure to treat early may result in death. Some sepsis definitions are based on the SIRS criteria, which include vital signs (e.g. heart rate and respiratory rate) and laboratory values suggestive of infection (e.g. white blood cell count). These definitions are referred to as Sepsis-1 and Sepsis-2 definitions. More recently, an international collaboration suggested a new definition based on end-organ dysfunction and the SOFA score, which is referred to as the Sepsis-3 definition (Singer et al. (2016) supra).

[0048] Methods of the invention may be used in conjunction with ICU scoring systems, such as sequential organ failure assessment (SOFA) score, Acute Physiology and Chronic Health Evaluation (APACHE) score (in particular, APACHE-II score), Simplified Acute Physiology Score (SAPS) (in particular, SAPS-II score), Charlson Comorbidity Index (CCI), and / or Glasgow Coma Score (GCS). Therefore, in one embodiment, the patient has a SOFA score of 9 or above, a SAPS II score of 59 or above and / or an APACHE II score of 22 or above. In a further embodiment, the patient has a SOFA score of 9-10, a SAPS II score of 59-63 and / or an APACHE II score of 22-25. VOL-C-P3782PCT

[0049] Methods of the invention may be performed for risk stratification to identify patients at risk or mortality (death). In one embodiment, the health care actions are prioritised based on the risk of mortality for the patient, determined from the level of nucleosomes measured in the sample.

[0050] Mortality may be caused by cardiovascular complications, lung diseases, lung embolism, thrombosis, thromboembolic complications, stroke, malignant diseases, sepsis, septic shock, bleeding disorders, organ failure, acute kidney disease, and others. The present invention allows for the prediction of the risk of mortality for the patient, particularly mortality due to sepsis.

[0051] References to “risk of mortality” means that patient is allocated either into a group of subjects of a population having a normal, i.e. non-elevated, risk for mortality or into a group of subjects having a significantly elevated risk. An elevated risk means that the risk of mortality within a predetermined predictive window (e.g. about 14 or about 28 days) is elevated significantly for a patient with respect to the average risk for mortality in a population of subjects. It is to be understood that the risk that shall be predicted is the short-term risk, i.e. the predictive window is short. In one embodiment, the risk of mortality within a period of up to 30 days is predicted. For example the risk of a poor outcome within a period of 3 days to 30 days, of 5 to 28 days, or of 7 to 14 days, is predicted. In particular, the predictive window is a period of about 28 days.

[0052] The inventors have found that a level of cell free nucleosomes (in particular, nucleosomes comprising histone isoform H3.1) greater than 20,000ng / mL indicates a critical condition with a very high mortality risk. For such levels, clinicians would be advised to act quickly. The inventors have found that a level of cell free nucleosomes (in particular, nucleosomes comprising histone isoform H3.1) greater than 10,000ng / mL indicates a very serious condition with about 25% mortality risk. The inventors have also found that a level of cell free nucleosomes (in particular, nucleosomes comprising histone isoform H3.1) greater than 1000ng / mL indicates the clinician should review other clinical parameters (such as urine output and / or platelet count). If there are signs of organ injury, treatment e.g. RRT, should be started.

[0053] In one embodiment, a level of nucleosomes greater than 20,000 ng / ml, such as greater than about 19000, 18000, 17000, 16000, 15000, 14000, 13000, 12000, 11000 or 10000 ng / ml indicates a high likelihood of patient mortality. It was found that these levels of nucleosomes presented a strong correlation with patients that died within about 14 days. In a preferred VOL-C-P3782PCT embodiment, a level of nucleosomes greater than 10,000 ng / ml indicates a high likelihood of patient mortality within 14 days.

[0054] In one embodiment, a level of nucleosomes greater than about 1 ,000 ng / ml indicates a high likelihood of patient mortality within 28 days. The level of nucleosomes may be greater than 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500 ng / ml. In a preferred embodiment, a level of nucleosomes greater than 6,800 ng / ml indicates a high likelihood of patient mortality within 28 days.

[0055] In one embodiment, the health care actions are prioritised based on the likelihood of an adverse outcome in the patient, determined from the level of nucleosomes measured in the sample. For example, an adverse outcome may include an acute coronary or cardiac event (such as a myocardial infarction and / or stroke), acute multi- or single-organ failure (such as renal failure, liver failure and / or heart failure), onset of a debilitating acute condition and / or an acute respiratory condition (such as pneumonia, hypoventilation / bradypnea, acute respiratory distress syndrome (ARDS) severe acute respiratory syndrome (SARS), bronchiolitis and / or bronchitis).

[0056] In one embodiment, the health care actions are prioritised based on the likelihood of kidney failure in the patient, determined from the level of nucleosomes measured in the sample.

[0057] In one embodiment, the kidney failure comprises acute kidney injury. Acute kidney injury (AKI, also known as acute renal failure, or ARF) is an abrupt (typically detected within about 48 hours to 1 week) reduction in glomerular filtration. Without quick treatment, abnormal levels of salts and chemicals can build up in the body, which affects the ability of other organs to work properly.

[0058] Methods of the invention may be performed for risk stratification to identify patients at risk for a future injury to kidney function, for future progression to reduced kidney function, for future progression to AKI, or for future improvement in kidney function.

[0059] The results herein indicate that a level of nucleosomes greater than 2489 ng / ml indicates a high likelihood of patient kidney failure within 28 days. The level of nucleosomes may be greater than about 2400, 2450 or 2500 ng / ml. In particular, the level of nucleosomes may be greater than 2480, 2490, 2500, 2510 or 2520 ng / ml. In one embodiment, a level of nucleosomes greater than about 2,500 ng / ml indicates a high likelihood of patient kidney failure within 28 days. VOL-C-P3782PCT

[0060] In one embodiment, the method comprises measuring one or more additional clinical parameters. The parameter(s) can be used in the interpretation of results.

[0061] Suitable clinical parameters may be selected from: level of platelets, urine output, level of creatinine, maximum serum lactate, level of urea, level of C Reactive Protein (CRP), level of proadrenomedullin, level of bilirubin, level of Procalcitonin (PCT), white blood cell count and level of albumin. Additional clinical parameters are referenced in Table 1 and Table 2 of the Examples.

[0062] Measurement of many of these parameters is already known in the art. For example, the level of platelets (also referred to as “platelet count” or “thrombocyte count”) may be determined manually using a hemocytometer or with an automated analyser. Thrombocytopenia is generally defined as a platelet count of less than 100x109 / L in an ICU setting.

[0063] As another example, in the ICU it is routine procedure to monitor urine output, such as every hour and / or every 24 hours (i.e. to calculate a mL per day value). The amount of urine produced is an indicator of fluid status and renal function. Generally, Foley catheters are connected to urine collection vessels that are inscribed with volumetric scales. These are used to manually observe and measure urine output at certain time intervals.

[0064] In a further embodiment, the clinical parameters are selected from maximum serum lactate, urine output, level of urea and / or level of platelets.

[0065] In one embodiment, the method additionally comprises measuring the urine output and / or level of platelets of the patient upon admission to hospital.

[0066] The results herein indicate that a level of platelets less than 84 x 109 / L indicates a high likelihood that the patient will require RRT within 28 days. The level of platelets may be less than about 100, 95, 90 or 85 x 109 / L. In one embodiment, a level of platelets less than about 84 x 109 / L indicates a high likelihood that the patient will require RRT within 28 days.

[0067] The results herein indicate that a urine output of less than 670 mL / day indicates a high likelihood that the patient will require RRT within 28 days. The urine output may be less than about 1000, 900, 800, 700, 690, 680 or 670 mL / day. In one embodiment, a urine output of less than about 670 mL / day indicates a high likelihood that the patient will require RRT within 28 days. VOL-C-P3782PCT

[0068] In one embodiment, a level of nucleosomes greater than 2,500 ng / mL, a level of platelets less than about 84 x 109 / L and a urine output of less than about 670 mL / day, indicates a high likelihood that the patient will require RRT within 28 days.

[0069] In one embodiment, a cumulative level of nucleosomes (in particular H3.1 -nucleosomes) greater than 12,400 ng / mL, a cumulative level of platelets less than about 171 x 109 / L and a cumulative urine output of less than about 694 mL / day, indicates a high likelihood that the patient will require RRT within 28 days.

[0070] Clinical parameters may include any relevant clinical information for example, demographic variables which include without limitation, gender, age, weight, height, Body Mass Index (BMI), smoking status and dietary habits. Therefore, in one embodiment, the clinical parameter is selected from the group consisting of: age, sex and BMI.

[0071] The clinical significance of a model comprising the level of nucleosomes and one or more clinical parameters, may be assessed using hazard ratios. The hazard ratio describes the relative risk of the complication (i.e. renal failure or mortality) based on a comparison of event rates. In one embodiment, a biomarker level described herein has a hazard ratio of less than 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, in particular less than 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, such as less than 4.1.

[0072] The sample may be any biological fluid (or body fluid) sample taken from a subject including, without limitation, cerebrospinal fluid (CSF), whole blood, blood serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

[0073] It will be understood that methods and uses of the present invention find particular use in blood, serum or plasma samples obtained from a patient. In one embodiment, the sample is a blood or plasma sample. Preferably, plasma samples are used. Plasma samples may be collected in collection tubes containing one or more anticoagulants such as ethylenediamine tetraacetic acid (EDTA), heparin, or sodium citrate, in particular EDTA. In an alternative embodiment, the sample is a serum sample. In a further embodiment both serum and plasma samples are used for the measurement of different members of an assay panel. VOL-C-P3782PCT

[0074] Measuring nucleosomes

[0075] The nucleosome is the basic unit of chromatin structure and 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 is wrapped approximately 146 base pairs of DNA. Another histone, H1 or H5, acts as a linker and is involved in chromatin compaction. The DNA is wound around consecutive nucleosomes in a structure often said to resemble “beads on a string” and this forms the basic structure of open or euchromatin. In compacted or heterochromatin this string is coiled and super coiled into a closed and complex structure (Herranz and Esteller, Methods Mol. Biol. (2007) 361 : 25-62).

[0076] 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.

[0077] It will be understood that the cell free nucleosome may be detected by binding to a component thereof. The term “component thereof’ as used herein refers to a part of the nucleosome, i.e. the whole nucleosome does not need to be detected. The component of the cell free nucleosomes may be selected from the group consisting of: a histone protein {i.e. histone H1 , H2A, H2B, H3 or H4), a histone post-translational modification, a histone variant or isoform, a protein bound to the nucleosome i.e. a nucleosome-protein adduct), a DNA fragment associated with the nucleosome and / or a modified nucleotide associated with the nucleosome. For example, the component thereof may be histone (isoform) H3.1 or histone H1 or DNA.

[0078] Methods and uses of the invention may measure the level of (cell free) nucleosomes per se. 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 per se may be measured by detecting a core histone protein, such as histone H4. As described herein, histone proteins form structural units known as nucleosomes which are used to package DNA in eukaryotic cells.

[0079] Normal cell turnover in adult humans involves the creation by cell division of a huge number of cells daily and the death of a similar number, mainly by apoptosis. During the process of apoptosis chromatin is broken down into mononucleosomes and oligonucleosomes which are released from the cells. Under normal conditions the levels of circulating nucleosomes found VOL-C-P3782PCT in healthy subjects is reported to be low. Elevated levels are found in subjects with a variety of conditions including many cancers, auto-immune conditions, inflammatory conditions, stroke and myocardial infarction (Holdenrieder & Stieber, Crit. Rev. Clin. Lab. Sci. (2009) 46(1): 1-24).

[0080] Previous nucleosome ELISA methods were used primarily in cell culture, usually as a method to detect apoptosis (Salgame et al., Nucleic Acids Res. (1997) 25(3): 680-681 ; Holdenrieder et al. (2001) supra, van Nieuwenhuijze et al., Ann. Rheum. Dis. (2003) 62: 10-14), but are also used for the measurement of circulating cell free nucleosomes in serum and plasma (Holdenrieder et al. (2001)). Cell free serum and plasma nucleosome levels released into the circulation by dying cells have been measured by ELISA methods in studies of a number of different cancers to evaluate their use as a potential biomarker.

[0081] The cell free nucleosome may be mononucleosomes, oligonucleosomes, a constituent part of a larger chromatin fragment or a constituent part of a NET or a mixture thereof.

[0082] Mononucleosomes and oligonucleosomes can be detected by Enzyme-Linked ImmunoSorbant Assay (ELISA) and several methods have been reported (e.g. Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)). These assays typically employ an anti-histone antibody (for example anti-H2B, anti-H3 or anti-H 1 , H2A, H2B, H3 and H4) as capture antibody and an anti-DNA or anti-H2A-H2B-DNA complex antibody as detection antibody.

[0083] 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. It will be clear to those skilled in the art that an elevation in nucleosome levels will 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). VOL-C-P3782PCT

[0084] In one embodiment, the component of the cell free nucleosome comprises an epigenetic feature of the cell free nucleosome. The level of nucleosomes may be measured by detecting an epigenetic feature of the nucleosome.

[0085] The biomarker used in the methods of the invention may be the level of cell free nucleosomes per se and / or an epigenetic feature of a cell free nucleosome. It will be understood that the terms “epigenetic signal structure” and “epigenetic feature” are used interchangeably herein. They refer to particular features of the nucleosome that may be detected. In one embodiment, the epigenetic feature of the nucleosome is selected from the group consisting of: a post- translational histone modification, a histone isoform, a modified nucleotide and / or proteins bound to a nucleosome in a nucleosome-protein adduct.

[0086] 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.

[0087] Nucleosomes measured in the present invention may contain the histone isoform H3.1. Therefore, in one embodiment, the histone isoform is H3.1. The inventors have established that H3.1 is not impacted by height, weight, age or sex of a patient or impacted by circadian rhythm, providing a suitable marker that is applicable across a range of patients.

[0088] 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 VOL-C-P3782PCT 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 present on a core nucleosome histone (e.g. H2A, H2B, H3 or H4), or a linker histone (e.g. H1 or H5). 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 and WO 2017 / 068359.

[0089] 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 methylation of a lysine residue. In a further embodiment, the methylation is of a histone 3 lysine residue. In a yet further embodiment, the histone PTM is selected from H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3 or H3K36Me3. In one embodiment, the histone PTM is acetylation of a lysine residue. In a further embodiment, the acetylation is of a histone 3 lysine residue. In a yet further embodiment, the histone PTM is selected from H3K9Ac, H3K14Ac, H3K18Ac or H3K27Ac. In another embodiment, the histone PTM is H4PanAc. In one embodiment, the histone PTM is phosphorylation of a serine residue. In a further embodiment, the phosphorylation is of an isoform X of histone 2A (H2AX) serine residue or phosphorylation of a histone 3 serine residue. In a yet further embodiment, the histone PTM is selected from pH2AX or H3S10Ph. In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is citrullinated H3 (H3cit) or citrullinated H4 (H4cit). In a further embodiment, the histone PTM is citrullination of a histone 3 arginine residue. In a yet further embodiment, the histone PTM is H3R8Cit. In one embodiment, the histone PTM is selected from the group consisting of: H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3, H3K36Me3, H3K9Ac, H3K14Ac, H3K18Ac, H3K27Ac, H4PanAc, pH2AX, H3S10Ph and H3R8Cit.

[0090] In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). In a yet further embodiment, the histone PTM is H3cit.

[0091] In one embodiment, the histone PTM is ribosylation, also referred to as ADP-ribosylation.

[0092] Post-translational histone ADP-ribosylation of nucleosomes occupying promoters of VOL-C-P3782PCT inflammatory response markers in macrophages is stimulated by exposure to lipopolysaccharides leading to elevated transcription and may have antiviral properties. Therefore, altered levels of circulating ADP-ribosylated nucleosomes released from macrophages are expected to be useful in methods of the invention.

[0093] 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 would include, for illustrative purposes without limitation, anti-pan-acetylation antibodies (e.g. a Pan-acetyl H4 antibody [H4panAc]), anti-citrullination antibodies or antiubiquitin antibodies.

[0094] In one embodiment, the epigenetic feature of the nucleosome comprises one or more DNA modifications. In addition to the epigenetic signalling mediated by nucleosome histone isoform and PTM composition, nucleosomes also differ in their nucleotide and modified nucleotide composition. Some nucleosomes may comprise more 5-methylcytosine residues (or 5- hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

[0095] In one embodiment, the epigenetic feature of the nucleosome comprises one or more proteinnucleosome 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.

[0096] Another way the structure of nucleosomes may vary is by mutation. Therefore, in one embodiment, the epigenetic feature is a mutated histone. In a further embodiment, the mutation is in histone 3 (H3). In a yet further embodiment, the mutation in H3 is when lysine 27 is replaced by a methionine (H3K27M). VOL-C-P3782PCT

[0097] In one embodiment, the protein adducted to the nucleosome (and which therefore may be used as a biomarker) is selected from: a transcription factor, a High Mobility Group Protein or chromatin modifying enzyme. References to “transcription factor” refer to proteins that bind to DNA and regulate gene expression by promoting ( / .e. activators) or suppressing ( / .e. repressors) transcription. Transcription factors contain one or more DNA-binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. All of the circulating nucleosomes and nucleosome moieties, types or subgroups described herein may be useful in the present invention.

[0098] It will be understood that more than one epigenetic feature of cell free nucleosomes may be detected in methods and uses of the invention. Multiple biomarkers may be used as a combined biomarker. Therefore, in one embodiment, the method comprises measuring more than one epigenetic feature of cell free nucleosomes as a combined biomarker. The epigenetic features may be the same type (e.g. PTMs, histone isoforms, nucleotides or protein adducts) or different types (e.g. a PTM in combination with a histone isoform). For example, a post- translational histone modification and a histone variant may be detected ( / .e. more than one type of epigenetic feature is detected). Alternatively, or additionally, more than one type of post-translational histone modification is detected, or more than one type of histone isoform is detected. In one aspect, the method comprises measuring a post-translational histone modification and a histone isoform as a combined biomarker in a sample, for the diagnosis, detection, treatment selection, prognostication or monitoring of a condition. In one embodiment, the combined biomarker is H3.1 and H3cit. In an alternative embodiment, the combined biomarker is H3.1 and H4cit.

[0099] Additional biomarkers

[0100] The level of cell free nucleosomes may be detected or measured as one of a panel of measurements.

[0101] A number of proteins occur in neutrophil extracellular traps (NETs). These proteins include, without limitation, myeloperoxidase (MPO), neutrophil elastase (NE), lactotransferrin, azurocidin, cathepsin G, leukocyte proteinase 3, lysozyme C, neutrophil defensin 1 , neutrophil defensin 3, myeloid cell nuclear differentiation antigen, S100 calcium-binding protein A8, S100 calcium-binding protein A9, S100 calcium-binding protein A12, actin p, actin y, alpha-actin, plastin-2, cytokeratin-10, catalase, alpha-enolase and transketolase (Urban et al., PLOS Pathogens. (2009) 10: e1000639). VOL-C-P3782PCT

[0102] In one embodiment, the level of nucleosomes may be detected or measured in combination with one or more markers of NETs. In one embodiment, the level of NETs is measured by measuring the level of neutrophil elastase, myeloperoxidase, C reactive protein, procalcitonin (PCT), D-Dimer and / or factor Vll-activating protease (FSAP) in the body fluid sample. In a further embodiment, the level of NETs is measured by measuring the level of neutrophil elastase or myeloperoxidase.

[0103] Biomarkers useful in a panel test include, without limitation, cytokine moieties (particularly interleukins), CRP, myeloperoxidase, neutrophil elastase, PCT, D-Dimer, FSAP, fibrinogen and fibrin / fibri nogen breakdown products. In one embodiment, the panel comprises one or more cytokines, such as one or more interleukins.

[0104] In one embodiment, the panel comprises C-reactive protein (CRP). CRP is a pentameric protein found in plasma and levels of CRP (whether or not adducted to nucleosomes) increase in plasma in response to inflammation, such as in bacterial, viral, fungal and microbial infections. CRP levels increase following IL-6 secretion by macrophages and T cells and its physiological role is to bind lysophosphatidylcholine expressed on the surface of dead or dying cells in order to activate the complement system via C1q. It also binds to phosphocholine on the surface of some bacteria and enhances phagocytosis. The measurement of CRP levels is useful for determining the progression of a condition and the effectiveness of treatments and elevated CRP levels have been shown in patients with increased risk of diabetes, hypertension and cardiovascular condition. Increased CRP levels have also been found in patients with kidney failure and inflammatory bowel condition (IBD, including Crohn’s condition and ulcerative colitis) and roughly correlate with coronary heart condition, although as elevated CRP is not directly related to heart condition it is not a specific prognostic marker. CRP may also be adducted to nucleosomes in NETs and nucleosome-CRP adduct is therefore a useful adduct for the detection of elevated levels of NETs in methods of the invention.

[0105] In one embodiment, the panel comprises myeloperoxidase. MPO is expressed in neutrophil granulocytes and produces hypohalous acids to carry out their antimicrobial activity. It is stored in azurophilic granules and released into the extracellular space during degranulation. The levels of MPO have been shown to be a useful predictor for myocardial infarction and have been combined with measurement of CRP for increased accuracy in predicting myocardial infarction risk in patients. In one embodiment, the panel comprises neutrophil elastase.

[0106] In one embodiment, the level of nucleosomes may be detected or measured in combination with one or more markers of AKI. In one embodiment, the one or more markers of AKI selected VOL-C-P3782PCT from: alanine aminopeptidase, y-glutamyl transpeptidase, calprotectin, cystatin C, tissue inhibitor of metalloproteinases-2 (TIMP-2), alkaline phosphatase (ALP), chitinase 3-like protein 1 , C-C motif chemokine ligand 14, hepcidin, Dickkopf-3, a glutathione S-transferase (GSTA), n glutathione S-transferase (GSTP), hepatocyte growth factor, insulin-like growth factor binding protein-7 (IGFBP7), kidney injury molecule-1 (KIM-1), neutrophil gelatinase- associated lipocalin (NGAL), netrin-1 , osteopontin, retinol binding protein, liver-type fatty acidbinding protein, N-acetyl-p-D-glucosaminidase, glomerular filtration rate, monocyte chemoattractant peptide-1 , proenkephalin A, serum creatinine, interleukin-18, urine output, tumour necrosis factor and / or microRNA in the body fluid sample. Measuring the level of nucleosomes in combination with the level of one or more of the markers of AKI can be used to establish the level of AKI.

[0107] In one embodiment, the method (e.g. for measuring the level of AKI) is used in conjunction with an imaging technique, such as ultrasound, doppler and computed tomography (CT) scanning. In a further embodiment, methods of the invention may be used in conjunction with Kidney Disease: Improving Global Outcomes (KDIGO) criteria.

[0108] The panel may also comprise different epigenetic features of the nucleosome as described hereinbefore (e.g. a histone isoform and a PTM).

[0109] Models can be derived using the biomarkers of the invention. Methods for deriving models or algorithms are well known in the art and suitable software packages are available. Typical software tools for this purpose include SPSS (Statistical Package for the Social Sciences) and “R”. These software packages provide for linear and non-linear data modelling of clinical data.

[0110] It will be clear to those skilled in the art, that any combination of the tests and biomarkers disclosed herein may be used in panels and algorithms for the detection or prediction of a complication to the condition, and that further markers may be added to a panel including these markers.

[0111] According to an aspect of the invention there is provided the use of a panel test to manage the clinical pathway for a patient admitted to hospital, comprising reagents suitable for determining levels of a panel of biomarkers, wherein the biomarkers comprise NETs, urine output and platelets. In one embodiment, the NETs are detected by determining the level of nucleosomes. In one embodiment, the biomarkers are measured in a body fluid sample obtained from the patient upon admission to hospital. VOL-C-P3782PCT

[0112] In one embodiment, the level of NETs is measured by measuring the level of nucleosomes, neutrophil elastase, myeloperoxidase, C reactive protein, procalcitonin, D-Dimer and / or factor Vll-activating protease in the body fluid sample.

[0113] The term “biomarker” as used herein, means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.

[0114] Biomarkers are also useful as companion diagnostic products for the selection of patients suitable for treatment by a particular therapy.

[0115] Measuring, Diagnosis and Monitoring Methods

[0116] The methods of the invention are directed to assigning the patient with a clinical pathway upon admission to hospital. The aim of the invention is to prioritise and manage patients based on their likelihood of an adverse outcome including mortality and / or an acute event requiring immediate medical care, for example surgery.

[0117] It will be understood that methods of the invention may also be used to identify patients who do not require hospital treatment, i.e. using the level of cell free nucleosomes detected to determine if the patient can be discharged from emergency care units, such as the ICU. This mode of the invention would help to identify patients who can be discharged early.

[0118] In one embodiment, circulating nucleosome levels are measured in a sample taken from a subject suffering from sepsis or septic shock, to determine the prognosis of the condition. Further measurements on multiple samples taken at intervals from a patient suffering from sepsis or septic shock may be made to monitor the progress of the condition and / or to assess the efficacy of treatment.

[0119] Identifying individuals at high risk of severe reaction or a complication, including organ failure, would allow triaging and facilitate allocation of strained medical resources. Therefore, in an aspect of the invention, there is provided a method of determining the clinical pathway for a patient admitted to hospital, comprising:

[0120] (i) providing a body fluid sample obtained from the patient upon admission to hospital; VOL-C-P3782PCT

[0121] (ii) contacting the body fluid sample with an antibody that specifically binds histone H3.1 ; and

[0122] (iii) measuring the level of nucleosomes present in the body fluid sample by detecting the level of binding of the antibody that specifically binds histone H3.1 ; wherein the level of nucleosomes measured in the body fluid sample is used to determine one or more health care actions that form the clinical pathway for the patient.

[0123] If the method is used for monitoring the severity or progression of a disorder, this can inform the healthcare team whether the patient should be managed on to the next stage of the clinical pathway or whether the clinical pathway should be amended / changed. In this embodiment, the method may additionally comprise:

[0124] (i) repeating the detection or measurement of the level of nucleosomes in a body fluid obtained from the patient on one or more occasions;

[0125] (ii) using any changes in the level nucleosomes to determine whether there is progression along the clinical pathway or manage any changes to the clinical pathway for the patient.

[0126] According to a further aspect of the invention, there is provided the use of a level of nucleosomes in a body fluid sample as a biomarker, to manage the clinical pathway for patients upon admission to hospital.

[0127] According to a further aspect of the invention, there is provided a method of predicting a risk of mortality for a patient admitted to hospital, comprising:

[0128] (i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and

[0129] (ii) determining the risk of mortality of the patient based on the level of nucleosomes measured in the sample.

[0130] According to a further aspect of the invention, there is provided the use of a ligand or binder that specifically measures the level of nucleosomes in a body fluid sample, in the manufacture of a kit for use in a method of predicting a risk of mortality of the patient within 28 days of admission to hospital.

[0131] As described herein, a level of nucleosomes (such as nucleosomes comprising histone H3.1) greater than 10,000 ng / ml indicates a high likelihood of patient mortality within 14 days. In another embodiment, a level of nucleosomes (such as nucleosomes comprising histone H3.1) VOL-C-P3782PCT greater than 1 ,000 ng / ml (in particular, greater than 6,800 ng / ml) indicates a high likelihood of patient mortality within 28 days.

[0132] According to a further aspect of the invention, there is provided a method of determining the likelihood of a patient requiring renal replacement therapy (RRT) comprising measuring the level of neutrophil extracellular traps (NETs) in a body fluid sample obtained from the patient.

[0133] According to a further aspect of the invention, there is provided the use of a level of NETs in a body fluid sample as a biomarker, to identify patients with a high likelihood of requiring RRT within 28 days of admission to hospital.

[0134] According to a further aspect of the invention, there is provided the use of a ligand or binder that specifically measures the level of NETs in a body fluid sample, in the manufacture of a kit for use in a method of identifying patients with a high likelihood of requiring RRT within 28 days of admission to hospital.

[0135] Detecting and / or quantifying may be performed directly on the purified or enriched nucleosome sample, or indirectly on an extract therefrom, or on a dilution thereof. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Uses and methods of detecting, monitoring and of diagnosis according to the invention described herein are useful to confirm the existence of a condition, to monitor development of the condition by assessing onset and progression, or to assess amelioration or regression of the condition. Uses and 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.

[0136] The detection or measurement may comprise an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method. In particular, detection and / or measurement may comprise a 2-site immunoassay method for nucleosome moieties. 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-DNA modification or anti-adducted protein detection binding agent. Also, detection and / or measurement may comprise a 2-site immunoassay, for example employing combinations of a labelled or immobilized: anti-nucleosome, anti-histone modification, anti-histone variant / isoform, anti-DNA modification or anti-adducted protein binding agent. VOL-C-P3782PCT

[0137] The immunoassays described herein for the measurement of nucleosomes, e.g. H3.1- nucleosomes or citrullinated nucleosomes, use high avidity and specificity monoclonal antibodies for binding to nucleosomes and NETs. These antibodies bind strongly and specifically to NETs, NETs metabolites and nucleosomes.

[0138] The inventors herein used a 2-site immunoassay for H3.1 -nucleosomes employing an immobilized anti-histone H3.1 antibody directed to bind to an epitope around amino acids SO- 33 of the histone H3.1 protein to capture clipped and non-clipped 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.

[0139] In one embodiment, the antibody specifically binds to an epitope present in histone H3.1 (an “anti-histone H3.1 antibody”). The amino acid sequence of histone H3.1 is known in the art and is described at UniProt Accession No. P68431. In mammals, histone H3 variants include: H3.1 , H3.2, H3.3, H3t and Centromeric Protein A (CENP-A). Human H3.1 and H3.2 are 99% identical (differing by only one amino acid), whereas histone H3t is 96% identical to H3.1 (differing at four amino acid positions) and histone H3.3 is 96% identical to H3.1 (differing at five amino acid positions). CENP-A shares only 46% identity with H3.1. In one embodiment, the antibody selectively binds histone H3.1 , H3.2 and H3t. It will be understood in this embodiment, the antibody does not bind to other histone H3 isoforms, such as histone H3.3 and CENP-A.

[0140] Nucleosomes are subject to clipping in which the histone tail is physically and irreversibly removed by regulated proteolysis, or clipping. Furthermore, histone degradation has been shown to be involved in the formation of NETs (see Papayannopoulos et al. (2010) J. Cell Biol. 191 (3): 677-691). On histone H3, clipping is reported to occur around amino acid position 21 (Yi and Kim (2018) BMB Reports, 51(5): 211-218). Therefore, in one embodiment, the antibody specifically binds to an epitope located higher than amino acid position 21. This enables capture of both clipped and non-clipped nucleosomes.

[0141] The amino acid sequence of histone H3.1 at positions 27-36 is KSAPATGGVK (SEQ ID NO: 1). The amino acid sequence at positions 29-35 does not include any commonly post- translationally modified amino acids (for example lysine, serine or arginine). Therefore, antibodies directed to bind to this epitope (i.e. amino acid positions 29-35) are unaffected, or minimally affected, by the post-translational modification status of the nucleosome, and will bind to all or most nucleosomes containing histone H3.1 , regardless of PTM structure. In one VOL-C-P3782PCT embodiment, the anti-histone H3.1 antibody specifically binds to an epitope comprising amino acids 30-33 (PATG, SEQ ID NO: 2) of histone H3.1. In another embodiment, the anti-histone H3.1 antibody specifically binds to an epitope comprising amino acids 28-32 (SAPAT, SEQ ID NO: 3) of histone H3.1. In particular the epitope is in or around amino acid 31 of histone H3 which is an alanine in H3.1 , but a serine in H3.3. Binding in these regions ensures that both intact and clipped nucleosomes are captured by the antibody regardless of their PTM status. This maximises the capture of H3.1 -nucleosomes.

[0142] In one embodiment, the anti-nucleosome antibody specifically binds to an epitope present in intact nucleosomes. The antibody does not bind (or binds weakly) to free histone octamer complexes, free histones (i.e. without DNA), free DNA or free histones. Again, the antibody may be relatively unaffected by the histone PTM composition of the nucleosomes to be bound. 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.

[0143] Therefore, in one embodiment of the invention the antibody is directed to bind to a conformational nucleosome epitope present in intact nucleosomes containing a histone octamer core complexed with DNA.

[0144] H3R8Cit nucleosome measurements may be performed using a 2-site immunoassay employing an immobilized antibody directed to bind to nucleosomes citrullinated at arginine 8 of histone H3, together with the same labelled anti-nucleosome antibody directed to bind to a conformational nucleosome epitope described hereinbefore.

[0145] 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.

[0146] In one 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; (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.

[0147] In another embodiment, the method of detection or measurement comprises: (i) contacting the sample with a first binding agent which binds to cell free nucleosomes; (ii) contacting the VOL-C-P3782PCT sample bound by the first binding agent in step (i) with a second binding agent which binds to an epigenetic feature of the cell free nucleosome; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

[0148] 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, a structural / shape mimic of the nucleosome or component part thereof. The term “measuring” or “determining” the amount of a biomarker as referred to herein refers to the quantification of the biomarker, e.g. to determining the level of the biomarker in the sample, employing appropriate methods of detection, as described herein.

[0149] 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, oligonucleosomes, NETs 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.

[0150] The antibodies used by the inventors for immunoassay are generally mouse monoclonal antibodies.

[0151] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen -binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise VOL-C-P3782PCT modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri- scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0152] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., I gG 1 , 1 gG2, 1 gG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively.

[0153] As used herein, the term “binding” or "specific binding" refers to the specificity of a binder, e.g., an antibody, such that it preferentially binds to a target, such as a polypeptide antigen. When referring to a binding partner, e.g., protein, nucleic acid, antibody or other affinity capture agent, etc., "specific binding" can include a binding reaction of two or more binding partners with high affinity and / or complementarity to ensure selective hybridization under designated assay conditions. Typically, specific binding will be at least three times the standard deviation of the background signal. Thus, under designated conditions the binding partner binds to its particular target molecule and does not bind in a significant amount to other molecules present in the sample. Recognition by a binder or an antibody of a particular target in the presence of other potential interfering substances is one characteristic of such binding. Preferably, binders, antibodies or antibody fragments that are specific for or bind specifically to a target bind to the target with higher affinity than binding to other non-target substances. Also preferably, binders, antibodies or antibody fragments that are specific for or bind specifically to a target avoid binding to a significant percentage of non-target substances, e.g., non-target substances present in a testing sample. In some embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred. For example, binders, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of non-target substances. In other embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non- target substances. VOL-C-P3782PCT

[0154] 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.

[0155] The term “detecting” or “diagnosing” as used herein encompasses identification, confirmation, and / or characterisation of a condition state. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a condition, to monitor development of the condition by assessing onset and progression, or to assess amelioration or regression of the condition. 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.

[0156] Methods of the invention may involve normalisation of marker levels. For example, the level of cell free nucleosomes containing a particular epigenetic feature may be normalised against the level of nucleosomes per se (or some other type of nucleosomes or parameter) to express the level as a proportion of nucleosomes containing the feature. For example, to express the level of citrullinated nucleosomes as the proportion of nucleosomes that are citrullinated.

[0157] Methods of the invention are preferably performed by measuring a marker level in a sample obtained upon admission to hospital (i.e. an “initial level”). Patients in ICU are monitored daily, therefore a marker level may also be established from samples obtained during the course of a patient’s stay in hospital, such as at days 0-7 after patient admission to hospital.

[0158] Methods of the invention may use the cumulative level of a marker. Such measurements taken over a period of time can support a real-time decision. It is calculated by combining several readings taken over a set time period. The accumulated level of a particular marker / parameter may be obtained by summing the area of each trapezoid calculated from the marker levels VOL-C-P3782PCT measured over a specified time period, e.g. upon admission to hospital (i.e. the initial level, day 0) and daily measurements taken over a 7 day period.

[0159] Using cumulative levels can avoid missing readings extrapolated between the first and last readings, as well as ignoring values which are inconsistent with a set minimum reference level. Therefore, measuring a cumulative level will often require a set minimum reference level in order to establish whether or not a single measurement should be included in the overall, cumulative level. The minimum reference level can be established using techniques known in the art and will vary depending on the marker to be measured.

[0160] A combination of initial and cumulative levels may be used for different markers described herein. For example, the method may comprise measuring the cumulative level of H3.1- nucleosomes (e.g. over 7 days after admission to hospital), in combination with the initial levels of platelet count and urine output.

[0161] In one embodiment, the method described herein is 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 condition state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration, relapse and / or remission.

[0162] In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control(s) and / or with one or more previous test sample(s) 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 biomarker(s) in test samples taken on different occasions.

[0163] A change in the level of the biomarker in the test sample relative to the level in 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 disorder or condition.

[0164] Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies, such as steroid treatment, and new therapies in human VOL-C-P3782PCT subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.

[0165] 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.

[0166] Diagnostic or monitoring kits (or panels) are provided for performing methods of the invention. Such kits will suitably comprise one or more ligands for detection and / or quantification of the biomarker according to the invention, and / or a biosensor, and / or an array as described herein, optionally together with instructions for use of the kit.

[0167] According to a further aspect of the invention, there is provided a kit comprising reagents for use in the method as defined herein. In particular, the kit may comprise one or more reagents and / or devices suitable for measuring the parameters described herein. According to a further aspect, there is provide the use of a kit comprising one or more reagents to detect or measure the level of nucleosomes, to manage the clinical pathway for a patient.

[0168] According to a further aspect of the invention, there is provided a kit for detecting a panel of biomarkers in a body fluid sample, comprising reagents suitable for determining levels of the panel of biomarkers, wherein the biomarkers comprise NETs, urine output and platelets; optionally comprising instructions for use of the kit in the method described herein. As described herein, the level of NETs may be detected by determining the level of nucleosomes in a body fluid sample.

[0169] A further aspect of the invention is a kit for managing the clinical pathways for a patient comprising a biosensor capable of detecting and / or quantifying one or more of the biomarkers as defined herein. As used herein, the term “biosensor” means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein. Biosensors may comprise a ligand binder or ligands, as described herein, capable of specific binding to the biomarker. Such biosensors are useful in detecting and / or quantifying a biomarker of the invention.

[0170] In one embodiment, the kit comprises one or more binding agents as described herein, for example a first binding agent which specifically binds to an epigenetic feature of a cell free nucleosome (e.g. H3.1) and a second binding agent which specifically binds to cell free nucleosomes. In one embodiment, the kit comprises reagents suitable for determining the level of nucleosomes, wherein the reagents comprise a 2-site immunoassay employing a VOL-C-P3782PCT labelled anti-nucleosome binding agent in combination with an immobilized anti-histone H3.1 binding agent. The immobilized binding agent may be present on a magnetic particle bead as the solid phase.

[0171] Suitably, biosensors for detection of one or more biomarkers combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal. Biosensors can be adapted for "alternate site" diagnostic testing, e.g. in the ward, outpatients’ department, surgery, home, field and workplace. Biosensors to detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic, Bio-Layer Interferometry (BLI) and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the one or more biomarkers.

[0172] 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, 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. In one embodiment, the immunoassay is a chemiluminescence immunoassay. The methods can employ a variety of label types including radioactive, enzyme, luminescent, fluorescent, time-resolved fluorescent and particulate labels. Output from the label can be detected using methods known in the art, such as by using an automated analyser, for example the IDS-i10 automated analyser system (Immunodiagnostic Systems Ltd, Boldon, UK). All of said immunoassay methods are well known in the art, see for example Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

[0173] 2-site immunoassays employed for methods of the invention may be one-step assays in which both antibodies and the sample are co-incubated in a single reaction step. Alternatively, two- step assays may be employed in which one antibody and the sample are co-incubated in a first reaction step, following which the sample is removed and bound analyte is exposed to the second antibody in a separate reaction step. VOL-C-P3782PCT

[0174] Identifying, detecting and / or quantifying can be performed by any method suitable to identify the presence and / or amount of a specific protein in a biological sample from a subject or a purification or extract of a biological sample or a dilution thereof. In particular, quantifying may be performed by measuring the concentration of the target in the sample or samples. Biological samples that may be tested in a method of the invention include those as defined hereinbefore. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. The present invention finds particular use in plasma samples which may be obtained from the subject.

[0175] The term “detecting” or “diagnosing” as used herein encompasses identification, confirmation, and / or characterisation of a disease state, degeneration state or health state of an individual or an organ. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the health or condition of a subject or an organ, to monitor development of the condition by assessing onset and progression, or to assess amelioration or regression of the condition. 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.

[0176] Identification, detection and / or quantification of biomarkers may be performed by detection of the biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N- terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. It is noted in particular that peptides of the same or related sequence to that of histone tails are particularly useful fragments of histone proteins.

[0177] For example, detecting and / or quantifying can be performed by one or more method(s) selected from the group consisting of: immunoassay, immunochromatography, SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spectrometry (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, LIPLC and other LC or LC MS-based 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.

[0178] Methods involving detection and / or quantification of one or more biomarkers of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, VOL-C-P3782PCT diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician’s office or at the subject’s bedside. Suitable biosensors for performing methods of the invention include “credit” cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-medicine. Therefore, in a further aspect of the invention, there is provided the use of a near patient or point-of-care immunoassay method for the measurement of a biomarker according to the invention. In one embodiment the near patient immunoassay method comprises a point-of-care immunoassay instrument (e.g. the Abbott i-STAT or the LightDeck Diagnostics point-of-care immunoassay instrument). In one embodiment the near patient immunoassay method comprises a lateral flow test. In a preferred embodiment the biomarker is a nucleosome or a nucleosome containing an epigenetic feature, in particular nucleosomes comprising histone H3.1.

[0179] The identification of biomarkers for a condition state permits integration of diagnostic procedures and therapeutic regimes. The biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels. The biomarkers may be used to provide warning of adverse drug response. Biomarkers are useful in development of personalized therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions. Thus, by monitoring a biomarker of the invention, subject care can be tailored precisely to match the needs determined by the disorder and the pharmacological profile of the subject, the biomarker can thus be used to titrate the optimal dose, predict a positive therapeutic response and identify those subjects at high risk of severe side effects.

[0180] Biomarker-based tests provide a first line assessment of ‘new’ subjects, and provide objective measures for accurate and rapid diagnosis, not achievable using the current measures.

[0181] Biomarker monitoring methods, biosensors, point-of-care tests, lateral flow tests and kits are also vital as subject monitoring tools, to enable the physician to determine whether relapse is due to worsening of the condition. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the condition, they provide an indication of the impact of drug therapy. VOL-C-P3782PCT

[0182] 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.

[0183] 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, sheep, goats, pigs, deer, llamas, cows and cattle.

[0184] The panels and methods described herein may be performed in vitro, or ex vivo. The methods described herein are preferably performed in vitro. 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.

[0185] 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 condition. For example, for conditions where the level of biomarker is higher in patients suffering from the condition, then if the level detected is higher than the cut-off, the patient is indicated to suffer from the condition. Alternatively, for conditions where the level of biomarker is lower in patients suffering from the condition, then if the level detected is lower than the cut-off, the patient is indicated to suffer from the condition. 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.

[0186] 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 condition or may be subjects with a different condition (for example, for the investigation of differential diagnosis). The “control” may comprise a healthy subject, and / or a subject without the condition. The control may also be a subject with the condition displaying no, or mild, symptoms. Mild symptoms may include manageable symptoms which do not require hospital intervention and / or intensive medical treatment.

[0187] In one embodiment, a subject who tests positive by methods of the invention may be afflicted with the condition and additionally suffers, or goes on to suffer, further medical complications. VOL-C-P3782PCT

[0188] In contrast, a control subject may also be afflicted with a condition but does not suffer, and does not go on to suffer, medical complications. Comparison with a control is well known in the field of diagnostics. The range of values found in the control group may be used as a normal or healthy or reference range against which the values found for test subjects can be compared. For example, if the reference range is <10 units, then a test value of 5 units would be considered normal, or not in need of treatment, but a value of 11 units would be considered abnormal and indicative of a need for treatment.

[0189] Therefore, in one embodiment, the method additionally comprises comparing the level of nucleosomes in the body fluid sample with one or more controls. For example, the method may comprise comparing the level of cell free nucleosomes present in a sample obtained from the subject with the level of cell free nucleosomes present in a sample obtained from a normal subject. The control may be a healthy subject.

[0190] In one embodiment, the level of nucleosomes is elevated compared to the control.

[0191] In one embodiment, comparison of the levels of the biomarkers in a body fluid sample with levels in the control samples is used to determine the health care actions for a patient. In one embodiment, the comparison is used to determine the risk of mortality for the patient. In another embodiment, the comparison is used to determine the likelihood of kidney failure in the patient.

[0192] It will be understood that it is not necessary to measure control levels for comparative purposes on every occasion. For example, for healthy 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 control subjects without the condition and testing for the level of biomarker. Results ( / .e. biomarker levels) for subjects suspected to have the condition 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 condition.

[0193] Methods of Treatment

[0194] According to a further aspect, there is provided a method of treating a patient admitted to hospital, which comprises the following steps:

[0195] (i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital;

[0196] (ii) using the level measured in step (i) to determine the health care actions for the patient; and VOL-C-P3782PCT

[0197] (iii) performing the health care actions determined in step (ii) in order to treat the patient.

[0198] Methods of the invention are performed on patients upon admission to hospital, in particular an ICU. Therefore, the health care actions may be one or more ICU medical interventions, as described hereinbefore. The intensive care treatment may comprise additional diagnostic procedures and therapeutic interventions. A therapeutic intervention may be a drug based therapy or comprises all kinds of surgical interventions.

[0199] As described herein, the method may comprise measuring one or more additional clinical parameters. For example, the method may also comprise measuring the maximum serum lactate, urine output, level of urea and / or level of platelets in the patient. The additional measurements can be combined with the level of nucleosomes measured to determine the health care actions for the patient.

[0200] The health care actions (i.e. therapy) may include one or more suitable treatments for the condition including without limitation, drugs (e.g. anti-inflammatory drugs, blood thinning or clotting inhibitor drugs, therapeutic anti-NETs antibody drugs, DNase drugs, NETosis inhibitor drugs, anti-bacterial drugs or anti-viral drugs), apheresis treatments, ventilator support, fluid support or others.

[0201] Preferably, said health care actions are selected from the medical guidelines or recommendations for management of sepsis such as International Guidelines for Management of Sepsis and Septic Shock (Evans et al. (2021) Intensive Care Med. 47(11): 1131-1247). In particular, the health care action may be treatment of sepsis or further diagnostic investigation or other aspects of care deemed necessary by the healthcare team.

[0202] The therapy may include one or more suitable treatments for the condition including e.g. anticoagulation drugs or anti-inflammatory drugs. Supportive treatments ay include plasma transfusions, transfusions of red blood cells and / or platelets, and anti-coagulant medication.

[0203] In one embodiment, the treatment is an anti-coagulant drug such as heparins, such as low weight heparins (LMWHs) and unfractionated heparin (UFH), warfarin, dabigatran, rivaroxaban, apixaban and edoxaban.

[0204] Treatments also include surgical removal and inferior vena cava filters. A patient may also be prescribed surgical stockings and exercise. VOL-C-P3782PCT

[0205] In one embodiment, the treatment is selected from one or more of: antibiotic treatments (e.g. penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides, clindamycin, sulphonamides, trimethoprim, metronidazole, tinidazole, quinolones and / or nitrofurantoin), anti-microbial treatments (e.g. ethambutol, isoniazid, pyrazinamide, rifampicin, aminoglycosides (amikacin, kanamycin), polypeptides (capreomycin, viomycin, enviomycin), fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), thioamides (ethionamide, prothionamide), cycloserine (closerin), terizidone, rifabutin, macrolides (clarithromycin), linezolid, thioacetazone, thioridazine, arginine, vitamin D and / or R207910), anti-viral COVID treatments (e.g. remdesivir), anti-viral influenza treatments (e.g. amantadine, umifenovir, moroxydine, rimantadine, umifenovir, zanamivir and neuraminidase inhibitors, capdependent endonuclease inhibitors, adamantanes, peramivir, zanamivir, oseltamivir phosphate and baloxavir marboxil) as well as anti-viral treatments for other viral diseases that may lead to a high level of NETosis and anti-fungal treatments (e.g. clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole and amphotericin).

[0206] In one embodiment, the treatment is an anti-inflammatory drug. Many steroidal and nonsteroidal anti-inflammatory drugs are known in the art. Some examples of steroidal antiinflammatory drugs include without limitation, dexamethasone, methotrexate, azathioprine, mycophenolate, cyclophosphamide, tocilizumab, rituximab, hydrocortisone, cortisone, betamethasone, prednisone, prednisolone, triamcinolone and methylprednisolone. Some examples of non-steroidal anti-inflammatory drugs include without limitation, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, CD24Fc (CD24 protein attached to the Fc region of immunoglobulin G) and EXO-CD24 (CD24-Exosomes).

[0207] In one embodiment, treatment is a DNase treatment to digest excess NETs or an inhibitor of NETosis, such as an anthracycline drug. In a further embodiment, the anthracycline drug is selected from: epirubicin, daunorubicin, doxorubicin and idarubicin.

[0208] The method may additionally comprise obtaining or determining the value from one or more ICU scoring systems. Such scoring systems include the sequential organ failure assessment (SOFA) score, the Acute Physiology and Chronic Health Evaluation (APACHE) score (in particular, APACHE-II score), the Simplified Acute Physiology Score (SAPS) (in particular, SAPS-II score), and / or the Charlson Comorbidity Index (CCI). The health care actions may be determined (i.e. to manage a clinical pathway) of a patient with (suspected) sepsis or septic VOL-C-P3782PCT shock based on both the ICU score and the results of the one or more biomarkers as described herein.

[0209] 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.

[0210] The invention will now be illustrated with reference to the following non-limiting examples.

[0211] EXAMPLES

[0212] EXAMPLE 1 - Association of circulating nucleosome biomarkers with mortality and kidney failure in the SISPCT Study

[0213] The randomised, placebo-controlled trial of Sodium Selenite and Procalcitonin Guided Antimicrobial Therapy in Severe Sepsis (SISPCT) was a multicentre clinical trial that evaluated the efficacy of high-dose intravenous sodium selenite treatment and procalcitonin-guided antimicrobial therapy in patients with severe sepsis or septic shock (Bloos et al. (2016) Effect of Sodium Selenite Administration and Procalcitonin-Guided Therapy on Mortality in Patients With Severe Sepsis or Septic Shock: A Randomized Clinical Trial. JAMA Intern Med 176:1266-1276). This large, well-characterised cohort provides an opportunity to further investigate the incidence and outcomes of renal failure in sepsis; 1089 patients with severe sepsis or septic shock were included in the intention-to-treat analysis. At baseline, 16.4% of patients required renal replacement therapy (RRT), with rates ranging from 13.2% to 22.2% across the different treatment groups.

[0214] Circulating H3.1-nucleosome levels in the serum samples from 971 patients who participated in the SISPCT study were analysed. The primary objective was to evaluate the potential of H3.1 -nucleosome levels as an early biomarker for sepsis and as a predictor of multiple organ failure and mortality. By leveraging this large, well-characterised cohort, the analysis provided insights into the diagnostic and prognostic value of H3.1 nucleosomes in sepsis management.

[0215] Methods

[0216] Study design and study population

[0217] This was a secondary biomarker analysis of serum levels of neutrophil extracellular traps using circulating nucleosomes in plasma samples collected from patients who participated in the multi-centre, prospective, randomised, placebo-controlled SISPCT trial (Clinicaltrials.gov identifier, NCT00832039). The study design and population has been described previously in detail (Bloos et al. (2016) supra). The trial was conducted from November 6, 2009, to June 6, VOL-C-P3782PCT

[0218] 2013, and included a 90-day follow-up period. Briefly, adults with severe sepsis or septic shock who were admitted to 33 ICUs across Germany were included and all patients were treated according to the Guidelines of the Germany Sepsis Society. For the analysis, 28-day survival and the development of kidney failure (any RRT, time to RRT initiation) were analysed. Kidney dysfunction was defined as urinary output of 0.5 ml / kg / h for at least 1 hour despite sufficient fluid resuscitation and / or increase of serum creatinine 2 above the reference range.

[0219] The study protocol was approved by the ethics board of Jena University Hospital and institutional review boards of all participating centres. The trial was carried out according to the Declaration of Helsinki and written informed consent was obtained from all patients or their legal representatives (World Medical Association (2013) World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310:2191-2194).

[0220] Blood sample collection

[0221] Blood samples were collected on days 0, 1 , 2, 3, 4, 5, 6, 7, 10, and 14, 21 , 28 after randomization if the patient was still in the ICU. Citrate plasma samples were stored at -80°C at the central study laboratory in Jena, Germany.

[0222] Nu.Q® NETs ImmunoAssay

[0223] Circulating nucleosomes containing H3.1 were measured using Nu.Q® NETs immunoassay (CE-IVDD, Belgian Volition SRL, Isnes, Belgium) according to manufacturer’s instructions. This sandwich immunoassay is based on chemiluminescence technology and was performed using the IDS-i10 automated analyser system (Immunodiagnostic Systems Ltd, Boldon, UK). Briefly, 50 pl of thawed citrate plasma were incubated with acridinium ester labeled antinucleosome detection antibody before magnetic particle beads, coated with the monoclonal anti-histone variant H3.1 were added. After a wash step, trigger solutions were added and the light emitted by the acridinium ester was measured via the luminometer system. The nucleosome concentration of each sample is automatically calculated by the immunoassay analyser using a 4-parameter logistic curve. All samples were analysed in duplicate. If the sample was above the limit of quantification and the coefficient of variation (%CV) of the determined concentration was above 20%, the analysis was repeated. Samples with concentrations >1200 ng / mL were automatically diluted to 1 :5 by the instrument; those with estimates of concentration >6000 ng / mL were manually pre-diluted 10 times before retesting. VOL-C-P3782PCT

[0224] Statistical analysis

[0225] We tested the primary and secondary hypotheses that serum NET levels are associated with 28-day mortality and commencement of RRT, respectively, in a large heterogenous group of critically unwell adult patients with sepsis.

[0226] Distribution of each variable were reviewed, and the imputations were performed for missing values of the SOFA and APACHE II scores, as per the SISPCT study statistical analysis plan. Continuous numerical variables were summarised using median values and categorical variables were summarised as counts and percentage of total. The range of initial H3.1- nucleosome serum levels, as a marker of NETs, was divided into quintiles. Patients receiving chronic intermittent dialysis, prior to their critical illness, were excluded from the analysis related to time to RRT outcome. We did not discriminate between continuous and intermittent modes of RRT.

[0227] Area under receiver operating characteristics (ROC) curves (ALICs) with 95% confidence intervals were calculated; Youden index was calculated to determine optimal cut-off points maximising sensitivity and specificity of continuous variables. Optimal cut-off points for continuous variables for survival curves were calculated using maximally selected rank statistics. Model AUC ROC curves and optimal cut-off points for continuous variables were determined.

[0228] Multi-variable time-to-event analysis for 28-day mortality and time-to-first RRT were conducted using Cox proportional hazards regression model. To compare the information value of the biomarker of interest, initial serum H3.1 -nucleosome levels, models were fit with and without the variable of interest and predictive accuracy compared. Variables were chosen using domain expertise in critically unwell sepsis patients.

[0229] All statistical analyses were performed using R 4.1.0 (R Core Team, Vienna, Austria) and survival, survminer and maxstat packages were used to analyse time-to-event data.

[0230] Results

[0231] Study population

[0232] Overall, 1089 patients were included in the SISPCT trial and data on clinical outcomes have previously been reported (Bloos et al. (2016) supra). Plasma samples from 971 patients were available for retrospective analysis. When applying the Sepsis 3 definition to patients included in the original study, 520 patients (53.6%) had septic shock and 443 (45.6%) had sepsis on admission. The median age of patients was 68 years and 65% were male. Overall, the VOL-C-P3782PCT baseline characteristics of the study population were representative of patients admitted to ICU with sepsis or septic shock (Table 1). Additional baseline data for clinical characteristics at admission are shown in Table 2. When grouped according to their initial levels of circulating H3.1 -nucleosomes being above (n=321) or below (n=549) a threshold of 1 ,000 ng / mL, those with H3.1 -nucleosomes >1 ,000 ng / mL had significantly more severe disease at baseline than those with values <1 ,000 ng / mL. This reflected the differentiation achieved when applying the Sepsis 3 definition, except for white blood cell counts and C-reactive protein (CRP) levels, which were significantly different between groups defined by their H3.1 -nucleosome levels (Table 1). Correlation analysis confirmed that H3.1-nucleosome levels were not influenced by patient age, height, sex or weight (data not shown).

[0233]

[0234]

[0235] ***p value <0.0001 ;aData are presented as median unless otherwise stated; initial values for circulating H3.1 -nucleosomes were only available for 870 patients;cMann-Whitney p value for sepsis vs septic shock population;dMann-Whitney p value for the H3.1- nucleosomes <1000 ng / mL vs H3.1 >1000 ng / mL populations.

[0236] APACHE II Acute Physiology and Chronic Health Evaluation II; BMI body mass index; CRP C reactive protein; ITU intensive treatment unit; SAPS II Simplified Acute Physiology Score II; SOFA Sequential Organ Failure Assessment.

[0237] VOL-C-P3782PCT

[0238] Table 2. Additional baseline clinical characteristics at admission VOL-C-P3782PCT VOL-C-P3782PCT

[0239] VOL-C-P3782PCT

[0240] Survival outcomes

[0241] Overall, 681 patients survived following admission to ICU. When analysed using a calculated cut-off point for overall survival of 1 ,143.3 ng / mL for H3.1-nucleosome levels at admission, a significant difference was observed between patients with H3.1 -nucleosome levels above or below this threshold (p=3.16 x 10'9; Fig. 1A). To establish whether H3.1 -nucleosome admission levels were associated with survival, cox regression analysis of 28-day mortality was performed. This revealed a negative association between high H3.1 -nucleosome levels and survival (Fig. 1 B). These data were also analysed by dividing the population equally into quintiles (Fig. 1C). We observed that among patients who die, all but two (9 out of 11) of those with H3.1-nucleosome levels >10,000 ng / mL died within 2 weeks (Fig. 2). This indicates that patients with H3.1-nucleosome levels >10,000 ng / mL at admission were at greater risk of death than patients with lower levels at admission.

[0242] To develop a predictive 28-day survival model, AUC analyses were used to determine the potential value of clinically relevant variables in the prediction of 28-day survival. Four variables, routinely used within the clinical setting as markers of infection severity, were identified alongside H3.1 -nucleosome levels. The model combined the following variables and calculated cut-off points: maximum serum lactate (12.9 mmol / L), urea (16 mmol / L), 24 h urine (1 ,035.8 mL), platelet count (102 x 109 / L) and H3.1-nucleosome levels (6,800 ng / mL). In AUC- ROC analysis the model performed well in predicting 28-day mortality (AUC of 71.86%) with best cut-off points with the Youden index identifying sensitivity of 76.11 % and specificity of 61.19% (Fig. 3).

[0243] Association with kidney failure

[0244] On admission, 33 patients were on chronic dialysis and information for time to RRT was missing for 11 patients, so these patients were excluded. Therefore, analysis was performed on data from 927 patients. Seven variables were identified and analysed individually for an association with kidney failure and the need for RRT using calculated cut points (Table 3). These included admission levels of H3.1 -nucleosomes, platelet count, creatinine, urea and C- reactive protein (CRP); 24 h urine; and maximum serum lactate. Of these, baseline 24 h urine, initial H3.1 -nucleosome levels and initial platelet counts were selected as having potential value in the identification of patients with sepsis or septic shock who were at risk of developing kidney failure and needing RRT. The calculated cut-off points for 28-day RRT were 670 mL and 84 x 109 / L for 24 h urine and platelet counts, respectively. As the cut-off point for H3.1- nucleosome levels can fall anywhere between 2488.5 and 2509.1 ng / mL, a standardised threshold of 2,500 ng / mL was applied. Individual ROC curves for each variable are presented in Fig. 4. VOL-C-P3782PCT

[0245] Table 3: Cut-off points calculated for 28-day RRT using the survminer R package

[0246] Scatterplots showed that combining H3.1 -nucleosome initial levels with either initial platelet counts or baseline 24 h urine can differentiate between patients who reguired either continuous RRT or intermittent dialysis and those who did not need RRT (Fig. 5A and B). When all 3 variables were combined for 28-day RRT using both cox regression analysis and a scoring model (Fig. 7) using the calculated cut-off points above, it revealed a significant differentiation between those with high versus low initial H3.1 -nucleosome levels. ROC-AUC analysis of the cox regression model showed that they performed well together to predict kidney failure (Fig. 6A).

[0247] As baseline 24 h urine and initial platelet counts are routinely used in the ICU setting, we chose to develop and test a clinical model that combined them with initial H3.1 -nucleosome levels to predict time to RRT. Cut-off points for each of the variables were calculated and four model groups defined (Table 4). When the clinical model was applied to 28-day RRT data, AUC-ROC analysis showed it performed well at identifying patients reguiring RRT with an AUC of 80.65% and optimal Youden associated cut-off point at 71 .62 % sensitivity at 79.33 % specificity (Fig. 6B). Cox regression analysis of the model showed a clear differentiation between groups (Fig. 7). When the H3.1 -nucleosome level variable was removed from the model the differentiation between the groups was reduced and accuracy for predicting outcome lost (Fig. 8).

[0248] Table 4. Clinical Model to predict days to RRT VOL-C-P3782PCT

[0249] In conclusion, the results demonstrate that high levels of H3.1 nucleosomes at admission are independently associated with mortality and progression to renal failure. These findings confirm the value of circulating H3.1 nucleosomes as a therapeutic target and prognostic biomarker for predicting mortality and organ dysfunction in patients with sepsis and septic shock. The clinical model combining H3.1 -nucleosome level with established markers that are routinely used in ICU, allows clinicians to identify patients at risk of AKI early on, providing opportunities to target interventions toward the higher risk patients and exclude individuals that may not benefit. This has the potential to improve short- and long-term outcomes for patients, support clinical decisions and reduce the cost burden.

Claims

VOL-C-P3782PCTCLAIMS1. A method of managing the clinical pathway for a patient admitted to hospital, comprising:(i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and(ii) determining the health care actions for the patient based on the level of nucleosomes measured in the sample.

2. The method of claim 1 , wherein the patient is admitted to an intensive care unit (ICU) of the hospital.

3. The method of any preceding claim, wherein the patient is suffering from, or is likely to be suffering from, sepsis or septic shock.

4. The method of claim 3, wherein the patient has a Sequential Organ Failure- Assessment (SOFA) score of 9 or above, a Simplified Acute Physiology Score (SAPS) II score of 59 or above and / or an Acute Physiology and Chronic Health Evaluation (APACHE) II score of 22 or above.

5. The method of any preceding claim, wherein the health care actions are prioritised based on the risk of mortality for the patient, determined from the level of nucleosomes measured in the sample.

6. The method of any preceding claim, wherein a level of nucleosomes greater than 10,000 ng / ml indicates a high likelihood of patient mortality within 14 days.

7. The method of any preceding claim, wherein a level of nucleosomes greater than 1 ,000 ng / ml (in particular, greater than 6,800 ng / ml) indicates a high likelihood of patient mortality within 28 days.

8. The method of any preceding claim, wherein the health care actions are prioritised based on the likelihood of kidney failure in the patient, determined from the level of nucleosomes measured in the sample.

9. The method of claim 8, wherein the kidney failure comprises acute kidney injury (AKI).VOL-C-P3782PCT10. The method of any preceding claim, wherein a level of nucleosomes greater than 2,500 ng / ml indicates a high likelihood of patient kidney failure within 28 days.11 . The method of any preceding claim, wherein the method comprises measuring one or more additional clinical parameters.

12. The method of claim 11 , wherein the clinical parameters are selected from maximum serum lactate, urine output, level of urea and / or level of platelets.

13. The method of any preceding claim, wherein the method additionally comprises measuring the urine output and / or level of platelets of the patient upon admission to hospital.

14. The method of any preceding claim, wherein the health care actions comprise one or more ICU medical interventions.

15. The method of claim 14, wherein the ICU medical intervention is selected from renal replacement therapy (RRT), invasive ventilation and / or vasopressors.

16. The method of any preceding claim, wherein a level of nucleosomes greater than 2,500 ng / ml, a level of platelets less than about 84 x 109 / L and a urine output of less than about 670 mL / day, indicates a high likelihood that the patient will require RRT within 28 days.

17. The method of any preceding claim, wherein the body fluid sample is a blood, serum or plasma sample.

18. The method of any preceding claim, wherein the level of nucleosomes is measured by detecting an epigenetic feature of the nucleosome.

19. The method of claim 18, wherein the epigenetic feature of the nucleosome is a histone isoform, such as a histone isoform of a core nucleosome, in particular a histone H3 isoform, such as H3.1.

20. The method of any preceding claim, wherein the level of nucleosomes is measured using an immunoassay, immunochemical, mass spectroscopy, chromatographic, chromatin immunoprecipitation or biosensor method.VOL-C-P3782PCT21. The method of any preceding claim, wherein the method of measuring the level of nucleosomes comprises contacting the sample with a solid phase comprising a binding agent that detects nucleosomes or a component thereof, and detecting binding to said binding agent.

22. The method of any preceding claim, wherein the method of measuring the level of nucleosomes comprises a 2-site immunoassay employing a labelled anti-nucleosome detection binding agent in combination with an immobilized anti-histone H3.1 binding agent.

23. The method of any preceding claim, wherein the method of measuring the level of nucleosomes comprises: (i) contacting the sample with a first binding agent which binds to an epigenetic feature of a nucleosome; (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent which binds to nucleosomes; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

24. The method of any preceding claim, additionally comprising comparing the level of nucleosomes in the body fluid sample with one or more controls.

25. A method of determining the clinical pathway for a patient admitted to hospital, comprising:(i) providing a body fluid sample obtained from the patient upon admission to hospital;(ii) contacting the body fluid sample with an antibody that specifically binds histone H3.1 ; and(iii) measuring the level of nucleosomes present in the body fluid sample by detecting the level of binding of the antibody that specifically binds histone H3.1 ; wherein the level of nucleosomes measured in the body fluid sample is used to determine one or more health care actions that form the clinical pathway for the patient.

26. A method of predicting a risk of mortality for a patient admitted to hospital, comprising:(i) measuring the level of nucleosomes present in a body fluid sample obtained from the patient upon admission to the hospital; and(ii) determining the risk of mortality of the patient based on the level of nucleosomes measured in the sample.

27. The method of claim 26, wherein a level of nucleosomes (such as nucleosomes comprising histone H3.1) greater than 10,000 ng / ml indicates a high likelihood of patient mortality within 14 days.VOL-C-P3782PCT28. The method of claim 26, wherein a level of nucleosomes (such as nucleosomes comprising histone H3.1) greater than 1 ,000 ng / ml (in particular, greater than 6,800 ng / ml) indicates a high likelihood of patient mortality within 28 days.

29. A method of determining the likelihood of a patient requiring renal replacement therapy (RRT) comprising measuring the level of neutrophil extracellular traps (NETs) in a body fluid sample obtained from the patient.

30. The method of claim 29, wherein the level of NETs is measured by measuring the level of nucleosomes, neutrophil elastase (NE) or myeloperoxidase (MPO) in the body fluid sample.

31. The method of claim 30, wherein a level of nucleosomes greater than 2,500 ng / ml indicates a high likelihood that the patient will require RRT within 28 days.

32. The method of any one of claims 29 to 31 , wherein the method comprises measuring one or more additional clinical parameters.

33. The method of claim 32, wherein the clinical parameters are selected from maximum serum lactate, urine output, level of urea and / or level of platelets.

34. The method of any one of claims 30 to 33, wherein a level of nucleosomes greater than 2,500 ng / ml, a level of platelets less than about 84 x 109 / L and a urine output of less than about 670 mL / day, indicates a high likelihood that the patient will require RRT within 28 days.

35. Use of a level of NETs in a body fluid sample as a biomarker, to identify patients with a high likelihood of requiring RRT within 28 days of admission to hospital.

36. A kit for detecting a panel of biomarkers in a body fluid sample, comprising reagents suitable for determining levels of the panel of biomarkers, wherein the biomarkers comprise NETs, urine output and platelets; optionally comprising instructions for use of the kit in the method of any one of claims 1 to 34.

37. The kit of claim 36, wherein the NETs are detected by determining the level of nucleosomes in a body fluid sample.

38. The kit of claim 36 or claim 37, wherein the kit comprises one or more control samples comprising predetermined levels of the panel of biomarkers.

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