4-hydroxynonenal in t cells and monocytes as a biomarker for early detection of sepsis

Abstract

The invention relates to the field of medicine and clinical diagnostics, more in particular to means and methods for detecting sepsis. Provided is a method for diagnosing sepsis in a subject, said method comprising: (a) detecting 4-HNE (4-hydroxy-2-nonenal) protein adducts in (i) CD14+ monocytes and / or (ii) CD3+ T cells in a sample obtained from the subject; (b) comparing the level of 4-HNE protein adducts in CD14+ monocytes and / or CD3+ T cells to that of a non-sepsis reference value; (c) providing a diagnosis wherein a reduced level of 4-HNE adducts in CD14+-monocytes and / or CD3+ T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis.

Description

[0001] P138908PC00

[0002] Title: Means and methods for early detection of sepsis.

[0003] The invention relates to the field of medicine and clinical diagnostics. More in particular, it relates to means and methods for detecting sepsis.

[0004] Sepsis is a hfe -threatening syndrome characterized by multiple organ failure triggered by infection.

[0005] Sepsis is a time -dep endent medical emergency that can be difficult to recognise. It results from an immune response to an infection that the body cannot control, which, if not diagnosed and treated promptly, can rapidly progress to organ failure, septic shock, and death. Mortality rates from sepsis range between 15% to 30% for sepsis and 40% to 70% for septic shock.

[0006] Sepsis is marked by excessive production of inflammatory cytokines and bactericidal reactive oxygen species (ROS). ROS-mediated lipid oxidation generates 4-hydroxynonenal (4-HNE), a highly reactive aldehyde that forms protein adducts, damages DNA, and modulates immune signalling. Under homeostatic conditions, the concentration of 4-HNE is between 0.05 pM and 15 pM in human blood and serum (Dwivedi et al., 2007; Parola et al., 1999). However, during oxidative stress, the concentration of 4-HNE can rise to more than 100 pM, which has a toxic effect on cells and can trigger apoptosis (Smathers et al., 2012). To prevent this, cells can reduce 4-HNE through alcohol dehydrogenase and aldose reductase, oxidize 4-HNE through aldehyde dehydrogenase, or conjugate it to glutathione through a spontaneous or glutathione-S-transferase (GSH)-catalyzed reaction (Sonowal & Ramana, 2019; Spitz et al., 1991).

[0007] 4-HNE adduct formation alters immune functions.

[0008] The clinical presentation of sepsis is highly variable depending on the etiology. Fever can be a first manifestation of an infection that can progress to sepsis, with pneumonia being the most common presentation leading to sepsis. The most common sites of infection are the respiratory, urinary, andgastrointestinal systems, as well as the skin and soft tissue. Symptoms can be subtle and misleading, mimicking and / or found in the presence of a number of different conditions such as flu, and this complicates its timely detection.

[0009] Early antibiotics and fluid resuscitation are the cornerstones of sepsis treatment, and delay in antibiotic administration significantly increases in-hospital mortality. Early antibiotic therapy is essential to improve clinical outcomes and should be given within one hour of suspected sepsis.

[0010] Vasopressor therapy is indicated when fluid resuscitation fails to restore adequate mean arterial pressure and organ perfusion.

[0011] Hence, early recognition of sepsis and timely initiation of treatment are critical to improve sepsis outcome. However, identifying sepsis remains a significant challenge, particularly in its early stages.

[0012] Methods known in the art for diagnosing sepsis include, for example, the method disclosed in WO2016 / 145425A1, which involves measuring the expression level of a panel of 11 biomarkers in a biological sample. This assay relies on the analysis of expression profiles, which are based on transcriptionally regulated events. However, these events typically take several hours to days to occur, making the method too slow for effective rapid intervention, particularly in emergency or critical care situations. Furthermore, diagnostic performance is still limited by heterogeneity among individuals. In addition, this approach requires complex equipment (e.g. fast RT-qPCR based approaches) which limit availability, particularly in out-of-hospital settings such as general practitioners’ offices.

[0013] Currently, no objective tools exist that can diagnose sepsis with sufficient accuracy. As a result, healthcare providers often rely on clinical judgment to decide whether to initiate sepsis therapy (e.g., broad-spectrum antibiotics,fluid administration) and admit a patient to the hospital with frequent checks of vital parameters.

[0014] The present inventors therefore aimed at providing a simple, reliable diagnostic method and test set-up to distinguish sepsis from non-severe infections. Ideally, the test should provide a reliable diagnosis within a few hours, preferably within 30-90 minutes, after a patient manifests symptoms that could be indicative of a (developing) sepsis. Furthermore, the diagnostic method and test can preferably be performed without the need for calibration and / or an external standard for comparison.

[0015] Considering the increased ROS production in sepsis patients (Brealey et al., 2002; Galley, 2011), they investigated the role of 4-HNE in sepsis and whether the severity of sepsis correlates with 4-HNE adduct levels. To that end, a study was performed using samples from post-hoc selected patients who had visited the emergency department (ED) who either developed sepsis with a severe outcome (i.e., ICU admission, death, or a rise in SOFA-score of two or more within 72 h), sepsis without a severe outcome, and a control group without sepsis.

[0016] It was surprisingly found that sepsis patients exhibit a significant increase in 4-HNE protein adducts in peripheral blood lymphocytes, whereas two major cytokine-producing cell types (T cells and monocytes), show the opposite trend, i.e. a reduction in the amount of 4-HNE protein adducts. The use of these parameters was recognized to provide a prospective biomarker for early sepsis. More specifically, a reduction in the level of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells was found to be an important parameter for detecting and diagnosing early sepsis. As is demonstrated herein below, whereas total 4-HNE protein adduct levels in the entire lymphocyte population are increased in sepsis patients comparedto control patients, this has only low diagnostic performance, with an area under the curve (AUG) of only 57.6% in the receiver operating characteristic (ROC) curve. In contrast, determining a reduction in 4-HNE protein adduct levels in CD3+lymphocytes according to the invention has a substantially better diagnostic performance with an AUG of 74.8%.

[0017] Because 4-HNE forms very stable adducts with protein, the time window for processing a blood sample after sampling is not critical. The 4-HNE protein adduct analysis can, therefore, be performed not only using fresh blood samples but also on freeze-thawed or (paraformaldehyde (PFA)) fixated blood cell samples. The diagnostic method can be readily implicated in or outside a standard clinical diagnostic setting, using, for example, flow cytometry or an immunochromatographic assay.

[0018] Herewith, the invention provides a non-invasive, specific, and rapid approach for diagnosing (early) sepsis in a subject.

[0019] Accordingly, in one embodiment, the invention provides a method for diagnosing sepsis in a subject, said method comprising:

[0020] (a) detecting 4-HNE (4-hydroxy-2-nonenal) protein adducts in (i) CD 14+ monocytes and / or (ii) CD3+ T cells in a sample obtained from the subject; (b) comparing the level of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells to that of a reference value; and

[0021] (c) providing a diagnosis, wherein a reduced level of 4-HNE adducts in CD 14+ monocytes and / or CD3+T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0022] Also provided is a method for diagnosing sepsis in a subject, said method comprising:(a) detecting 4-HNE (4-hydroxy-2-nonenal) protein adducts in (i) total peripheral blood leukocytes and in one or both of (ii) CD14+ monocytes and (iii) CD3+T cells in a sample obtained from the subject;

[0023] (b) calculating the ratio of 4-HNE protein adducts in CD 14+ monocytes to the 4-HNE protein adducts in peripheral blood leukocytes (“CD14 / total”) and / or the ratio CD3+ T cells to the 4-HNE protein adducts in peripheral blood leukocytes (“CD3 / total”) of the subject; and

[0024] (c) using the ratio(s) calculated under (b) to diagnosis whether or not the subject suffers from sepsis or is at risk of developing (severe) sepsis, preferably wherein a reduced ratio of CD14 / total and / or a reduced ratio of CD3 / total indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0025] A method of the invention is not taught or suggested in the art. There is ample prior art disclosing a correlation between increased 4-HNE protein adduct formation and inflammation, infection and sepsis. See for example Wu & Pan (2023), Am J Transl Res. 15: 1913-1920; Qiao, et al. (2023) Eur J Phar 950: 175702; Singh, et al. (2016). J Kidney. 2. 10.4172 / jok.l000116; Wiesel, et al. (2000) Circulation. 102: 3015-3022; Dalleau, et al. (2013) 20: 1615-1630.). It is also known that 4-HNE is not a specific biomarker for early sepsis itself, because other inflammatory diseases and metabolic disorders are also hallmarked by elevated ROS levels but not the respiratory burst.

[0026] However, in none of these studies an attempt was made to look at HNE adducts in certain subpopulations of cells. Even less so, the prior art suggests that (a) 4-HNE adducts are actually decreased in certain cell populations in sepsis patients and (b) the ratio of 4-HNE protein adducts in T cells and / or monocytes over neutrophils is a prospective biomarker for early sepsis is not disclosed or suggested in the art. Without wishing to bebound by theory, it is believed that the neutrophil-mediated ROS production in the respiratory burst causes more 4-HNE protein adduct formation in the neutrophils. Because ROS production also causes hypoxia (i.e., lack of oxygen), it concomitantly results in T cells and monocytes producing lower levels of 4-HNE.

[0027] In one aspect, a method of the invention comprises detecting 4-HNE protein adducts in (i) CD 14+ monocytes and / or (ii) CD3+T cells. In one aspect, it comprises detecting 4-HNE protein adducts in CD 14+ monocytes in a sample obtained from the subject; comparing the level of 4-HNE protein adducts in CD 14+ monocytes to that of a reference value; and providing a diagnosis wherein a reduced level of 4-HNE protein adducts in CD 14+ monocytes relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis. The reference value is obtained from non-sepsis control subjects. For example, the reference value is suitably obtained from healthy control subjects, non-infectious or infectious non-sepsis control subjects, preferably age- and gender-matched healthy or non-infectious or infectious non-sepsis control subjects.

[0028] Typically, a 10-30% or larger decrease (reduction) in the level of 4-HNE adducts in CD 14+ monocytes relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis. In one embodiment at least a 10%, 12%, 15%, 18%, 20%, 23%, 25%, 28% or 30% reduction in the level of 4-HNE protein adducts in CD 14+ monocytes relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis.

[0029] In one embodiment, a positive sepsis diagnosis is made based on a level of 4-HNE protein adducts in CD 14+ monocytes which is down to 90%, preferably down to 80%, down to 75%, down to 70%, down to 60% of a reference value.In another aspect, a method comprises detecting 4-HNE protein adducts in CD3+T cells in a sample obtained from the subject, comparing the level of 4-HNE protein adducts in CD3+T cells to that of a reference value; and providing a diagnosis wherein a reduced level of 4-HNE adducts in CD3+T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis. Typically, a 1.8 to 2-fold or more decrease (reduction) in the level of 4-HNE adducts in CD3+ T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis. For example, a patient sample showing at least a 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold or even higher reduction in the level of 4-HNE protein adducts in CD3+ T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis. Hence, whereas a reduction in the level of 4-HNE adducts is observed in both the CD 14+ monocyte and CD3+T cell populations of a sepsis patients, this reduction is typically most pronounced in the CD3+T cells.

[0030] In one embodiment, a positive sepsis diagnosis is made based on at least reduced level of 4-HNE protein adducts in CD3+ T cells, preferably wherein the CD3+T cell 4-HNE protein adduct level is 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, or 50% or less, compared to a reference value. In one aspect, a positive sepsis diagnosis is made based on a level of 4-HNE protein adducts in CD3+T cells in a range of 50-75%, 60-75%, 65-75%, 50-70%, 55-70% or 60-70% compared to a reference value.

[0031] In a preferred aspect, a method of the invention comprises (a) detecting 4-HNE protein adducts in both the CD 14+ monocyte and the CD3+ T cell populations in a sample obtained from the subject; (b) comparing the level of 4-HNE protein adducts in CD 14+ monocytes and CD3+T cells to that of a reference value; and (c) providing a diagnosis wherein a reduced level of 4-HNE protein adducts in CD 14+ monocytes and in CD3+T cells relative to thereference value indicate that the subject suffers from sepsis or is at risk of developing (severe) sepsis. Preferably, a diagnosis that a subject suffers from sepsis or is at risk of developing sepsis is based on at least a 10%, preferably at least 15% decrease in the level of 4-HNE protein adducts in CD 14+ monocytes and at least a 1.8-fold, preferably at least 2-fold decrease in the level of 4-HNE protein adducts in CD3+ T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis.

[0032] It is also possible to determine the absolute level of 4-HNE protein adducts in CD 14+ monocytes and / or (ii) CD3+T cells obtained from the subject, using e.g. a calibration curve of known amounts of 4-HNE protein adducts, and provide a diagnosis based on a significant reduction in the absolute 4-HNE protein adduct levels. Typical levels of 4-HNE protein adduct levels in CD 14+ monocytes and CD3+T cells of (human) healthy controls are between 1 and 10 pM, based on scientific literature (Dwivedi et al., 2007; Parola et al., 1999) and on calibration measurements where cells are treated with known concentrations of 4-HNE (see Figure 1).

[0033] Hence, in one embodiment the invention provides a method comprising determining the absolute level of 4-HNE protein adducts in (i) CD 14+ monocytes and / or (ii) CD3+T cells; and providing a diagnosis wherein a level of 4-HNE protein adducts in CD 14+ monocytes below 0.9 pM, preferably below 0.8 pM, more preferably below 0.7 pM and / or (ii) a level of 4-HNE protein adducts in CD3+ T cells below 0.6 pM, preferably below 0.5 pM, more preferably below 0.4 pM indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0034] In an alternative embodiment of the invention, a method also comprises detecting HNE protein adducts in total peripheral blood leukocytes in asample obtained from the subject. This allows to express the amount of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells of a (suspected) patient as the ratio of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells relative to the level of 4-HNE protein adducts in total peripheral blood leukocytes. An advantage of using the ratio of 4-HNE protein adducts in T cells and / or CD 14 monocytes (both decreased in sepsis; see Figures 4 and 5) over the total 4-HNE protein adduct levels (increased in sepsis; see Figure 3) is that a diagnostic method or test based on ratio parameter(s) does not require calibration and / or an external standard or reference value for comparison. It was found that the ratio of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells to the 4-HNE protein adducts in peripheral blood leukocytes, provides a preferred parameter for detecting early sepsis. More in particular, a ratio of CD14 / total below 2.7 and / or a ratio of CD3 / total below 1.4, indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis. In control individuals, these ratios are approximately 6.6 for both CD3 / total and CD14 / totals.

[0035] Suitably, a ratio of CD14 / total below 2.7, preferably below 2.6, more preferably below 2.5, such as below 2.4, 2.3, 2.2, 21 or even below 2.0 indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0036] For example, a ratio of CD3 / total below 1.4, preferably below 1.3, more preferably below 1.2, such as below 1.1 or even below 1.0 indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0037] Thus, a decrease in one or both these ratios, optionally combined with an increased absolute amount (level) of 4-HNE protein adducts in peripheral blood leukocytes, allows to distinguish between sepsis patients and patients suffering from an uncompromised (non-sepsis) infection with superior diagnostic performance.Accordingly, a method may comprise comparing the level of 4-HNE protein adducts in peripheral blood leukocytes of the subject to that of a reference value, wherein an elevated level relative to the reference value is a further indicator that the subject suffers from sepsis or is at risk of developing (severe) sepsis. In one embodiment, the level of 4-HNE protein adducts in peripheral blood leukocytes of the subject to that of a reference value, is at least a 6-fold, preferably at least a 7-fold, 8-fold, more preferably at least a 9-fold increase in 4-HNE protein adducts in peripheral blood leukocytes relative to the reference value.

[0038] In a preferred embodiment, the invention provides a method for diagnosing sepsis in a subject, said method comprising:

[0039] (a) detecting 4-HNE protein adducts in (i) total peripheral blood leukocytes and in one or both of (ii) CD 14+monocytes and (iii) CD3+T cells in a sample obtained from the subject;

[0040] (b) calculating the ratio of 4-HNE protein adducts in CD 14+ monocytes to the 4-HNE protein adducts in peripheral blood leukocytes (”CD14 / total”) and / or the ratio of 4-HNE protein adducts in CD3+T cells to the 4-HNE protein adducts in peripheral blood leukocytes (“CD3 / total”) of the subject; (c) comparing the level of 4-HNE protein adducts in peripheral blood leukocytes of the subject to that of a reference value, and

[0041] (d) using the information obtained under (b) and (c) to provide a diagnosis, wherein a ratio of CD14 / total below 2.7 and / or a ratio of CD3 / total below 1.4, combined with at least a 6-fold increase in the 4-HNE protein adducts in peripheral blood leukocytes indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

[0042] In a most preferred embodiment, the invention provides a method for diagnosing sepsis in a subject, said method comprising:(a) detecting 4-HNE protein adducts in (i) total peripheral blood leukocytes, (ii) CD 14+ monocytes and (iii) CD3+T cells in a sample obtained from the subject;

[0043] (b) calculating the ratio of 4-HNE protein adducts in CD 14+ monocytes to the 4-HNE protein adducts in peripheral blood leukocytes (”CD14 / total”) and the ratio of 4-HNE protein adducts in CD3+ T cells to the 4-HNE protein adducts in peripheral blood leukocytes (‘”CD3 / total”) of the subject; (c ) comparing the level of 4-HNE protein adducts in peripheral blood leukocytes of the subject to that of a reference value, and

[0044] (d) using the information obtained under (b) and (c) to provide a diagnosis, wherein a ratio of CD14 / total below 2.7 and a ratio of CD3 / total below 1.4, combined with at least a 6-fold increase in the 4-HNE protein adducts in peripheral blood leukocytes indicates that the subject suffers from sepsis, early stage sepsis or severe sepsis, or is at risk of developing (severe) sepsis.

[0045] As is demonstrated herein below, our data show that 4-HNE levels on peripheral blood leukocytes, CD3+ T cells and CD 14+ monocytes are advantageously used in combination with NEWS (National Early Warning Score) for recognition of a subject that will develop severe sepsis outcome (Figure 7 and 8).

[0046] The term “subject” as used herein refers to a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate. In a preferred embodiment, said subject is a human subject. Alternatively, the subject is an animal, like a husbandry animal, an equine animal, including a horse, or a household pet. Hence, the invention also has veterinary applications. The subject according to the invention can be a healthy subject or a subject suffering from a given disease such as sepsis.As used herein the term "Sepsis" means morbid condition induced by a toxin, the introduction or accumulation of which is caused by infection or trauma, and includes the early stage of sepsis, severe sepsis and the acute phase of septic shock.

[0047] The term “early stage sepsis” refers to the stage of the disease with the onset of clinical symptoms of severe infectious disease that typically include chills, profuse sweat, irregularly remittent fever, prostration and the like. The term “severe sepsis” refers to the stage of the disease with the clinical symptoms of early stage sepsis in addition to persistent fever, lymphopenia, disseminated intravascular coagulation, respiratory distress syndrome, multiple organ failure and hypotension leading to shock.

[0048] Suitably, the sample is a blood sample comprising leukocytes. For example, the sample is a huffy coat sample or a whole blood sample. In one aspect, a buffy coat sample of the subject is used to detect 4-HNE protein adducts in peripheral blood leukocytes, CD 14+ monocytes and / or CD3+T cells.

[0049] Total peripheral blood leukocytes refer to all white blood cells (WBCs) present in peripheral blood, including lymphocytes, monocytes, and granulocytes. These cells are essential for immune responses and include T cells, B cells, natural killer (NK) cells, monocytes, neutrophils, eosinophils, and basophils, each of which can be identified by specific CD (cluster of differentiation) markers. Lymphocytes, which make up ~70-90% of peripheral blood leukocytes, consist of T cells (CD3+, with CD4+for helper T cells and CD8+for cytotoxic T cells), B cells (CD19+, CD20+, CD21+), and natural killer (NK) cells (CD16+, CD56+). Monocytes (~2- 10%) express CD14+, CD16+, and CDllb+, while dendritic cells (<1%) include conventional DCs (CDllc+) and plasmacytoid DCs (CD123+). In some embodiments, a blood sample is collected after which the red blood cells are lysed prior to analysis to enrich for leukocytes.In research and clinical settings, peripheral blood leukocytes are often isolated from whole blood using Ficoll-Paque density gradient centrifugation, which enriches for peripheral blood mononuclear cells (PBMCs) — a subset of peripheral blood leukocytes that includes lymphocytes (T, B, NK cells) and monocytes, but excludes granulocytes and red blood cells. The term peripheral blood leukocytes is sometimes used interchangeably with PBMCs, but technically, peripheral blood leukocytes include all leukocytes, whereas PBMCs refer specifically to mononuclear cells without neutrophils or other granulocytes.

[0050] Monocytes are part of the mononuclear phagocyte system and are precursors to macrophages and dendritic cells. They circulate in the peripheral blood and can differentiate into various other immune cells when they migrate to tissues. The term ‘’CD14+monocytes” refers to a specific subset of monocytes, which are a type of white blood cell involved in immune responses. CD14+monocytes are characterized by the expression of the CD 14 marker, which is a co-receptor for lipopolysaccharide (LPS), a component of bacterial cell membranes. CD 14 is used as a surface marker to identify monocytes, and its expression is often used to distinguish these cells from other types of leukocytes in flow cytometry or immunohistochemistry. Monocytes can be subdivided into different subsets based on their expression of CD 14 and other markers (e.g., CD 16). Typically, CD14+monocytes are classified into (i) Classical monocytes: High CD 14 expression, low CD 16 expression; (ii) Non-classical monocytes: Low CD 14 expression, high CD 16 expression and (iii) Intermediate monocytes: Intermediate expression of both CD 14 and CD 16.

[0051] The term °CD3 T cells” refer to a subset of T lymphocytes that express the CD3 protein complex, which is a critical marker for all T cells. The presence of CD3 is used as a marker to identify and isolate all T cells from othertypes of immune cells, such as monocytes and B cells. The CD3 complex is part of the T-cell receptor (TCR) complex, which is essential for T cell activation and signaling. The CD3 protein complex is involved in transmitting signals from the TCR to the interior of the T cell after it recognizes an antigen, leading to the activation of the T cell. CD3+T cells include both CD4+T helper cells and CD8+cytotoxic T cells.

[0052] Methods for separating a sample obtained from a subject into the relevant cell populations (i.e., total peripheral blood leukocytes, PBMCs, CD 14+ monocytes, and / or CD3+T cells) can be achieved using methods known in the art. Typically, a sequential approach using density gradient centrifugation followed by magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) is used.

[0053] First, to isolate total peripheral blood leukocytes, whole blood can be collected in anticoagulant tubes (such as EDTA or heparin) and diluted 1:1 with phosphate-buffered saline (PBS). The diluted blood is carefully layered over Ficoll-Paque or another density gradient medium in a centrifuge tube and spun at 400-500xg for 30-40 minutes at room temperature without using the brake.

[0054] After centrifugation, the layer that contains leukocytes is collected from the interface between plasma and Ficoll. This interface, known in the art as huffy coat, is a thin layer of cells that forms between the plasma and the red blood cells (RBCs) when whole blood is subjected to density gradient centrifugation. It appears as a white or pale layer that sits above the red blood cells and below the plasma. The buffy coat contains a variety of white blood cells (leukocytes) and platelets. The cell types present in the buffy coat include lymphocytes (such as T cells, B cells, and natural killer cells), monocytes (which can differentiate into macrophages and dendritic cells),and granulocytes such as neutrophils, which are involved in combating infections, eosinophils, which play a role in allergic reactions and parasitic infections, and basophils, which are involved in inflammatory responses. Additionally, platelets, which are small cell fragments that contribute to blood clotting and tissue repair, are also found in the huffy coat.

[0055] The collected cells are then washed, e.g. with PBS by centrifugation at 300xg for 10 minutes, to remove platelets and resuspended in the desired medium (e.g., RPMI-1640 with 2% FBS).

[0056] For the detection of CD 14+ monocytes and / or CD3+ T cells by FACS, the total leukocyte fraction can be stained with the relevant (fluorophore-conjugated) antibodies i.e. anti-CD14 antibodies and / or anti-CD3 antibodies followed by processing using a flow cytometer, where CD14+cells and / or CD3+cells are gated and sorted. In a preferred embodiment, cells are stained using fluorochrome-conjugated antibodies.

[0057] A fluorochrome (or "fluorophore ”) is a chemical that can absorb energy from an excitation source (laser beam) and emit photons at a longer wavelength (fluorescence), which is captured by optical detectors of the flow cytometer. Suitable fluorochromes for conjugating antibodies are known in the art. Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser (s) used to excite the fluorochromes and on the detectors available. The fluorochromes are preferably selected for their brightness, limited spectral overlap, and limited need for compensation, stability, etc.

[0058] A wide range of fluorophores can be used as labels in flow cytometry. These fluorophores absorb light at a specific wavelength and emit it at a longerwavelength, which allows detection by the flow cytometer. The choice of fluorophore depends on factors such as the desired emission wavelength, the available lasers on the flow cytometer, and the specific experimental requirements.

[0059] Commonly used fluorophores in flow cytometry include FITC (Fluorescein isothiocyanate), which emits green fluorescence at around 525 nm when excited by a 488 nm laser, making it suitable for a variety of applications. Another widely used fluorophore is PE (Phycoerythrin), which emits orange fluorescence at around 575 nm when excited by a 488 nm laser. PE is brighter than FITC and often used for detecting low-abundance markers. APC (Allophycocyanin) emits far-red fluorescence at around 660-670 nm when excited by a 633 nm or 640 nm laser and is commonly used for experiments requiring a broader range of emission wavelengths. PerCP (Peridinin-chlorophyll-protein complex) emits red fluorescence at around 675 nm when excited by a 488 nm laser and is often used in tandem with other fluorophores for multicolor flow cytometry.

[0060] The Alexa Fluor series, including Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, and others, are a group of highly photostable and bright fluorophores with a wide range of emission spectra, making them useful for various multicolor flow cytometry experiments. PE-Cy5 and PE-Cy7 are tandem dyes that combine PE with other fluorophores, such as Cy5 or Cy7, providing additional emission wavelengths for multicolor experiments.

[0061] FITC-Cy5 and FITC-Cy7 work in a similar way, pairing FITC with Cy5 or Cy7 to allow additional detection channels in multicolor experiments.

[0062] BV421 (Brilliant Violet 421) emits violet fluorescence around 421 nm when excited by a 405 nm laser. It is part of the Brilliant Violet dye series, which are useful for multicolor flow cytometry, especially when using UV lasers. DAPI (4',6-diamidino-2-phenylindole) emits blue fluorescence when excited by UV light (around 350 nm) and is commonly used for staining DNA and assessing cell viability.KO525 is a relatively newer fluorophore used in flow cytometry. It is a green-fluorescent dye that is typically excited by a 488 nm laser and emits light in the range of approximately 525 nm. KO525 is similar to other green-fluorescent dyes, such as FITC or Alexa Fluor 488, but it is considered to have certain advantages, such as improved brightness, photostability, and reduced background fluorescence.

[0063] When choosing fluorophores for flow cytometry, it is important to ensure that the emission spectrum of the different fluorophores do not overlap significantly with other fluorophores being used in the experiment to avoid spectral overlap and improve resolution. Exemplary conjugated antibodies include anti-CD3-FITC, anti-CD3-PerCP, anti-CD14-KO525, anti-CD14-APC and functional equivalents thereof. In case both CD 14 and CD3 expressing subpopulations are detected using multicolor flow cytometry, it is preferred that the antibodies are conjugated to distinct fluorophores with minimal spectral overlap. For example, cells are stained with anti-CD14-KO525 antibodies and / or anti-CD3-PerCP antibodies the

[0064] Alternatively, for the isolation of CD14+monocytes by MACS, the total leukocyte or PBMC fraction is incubated with anti-CD14 magnetic beads (following the manufacturer’s protocol, typically for 15 minutes at 4°C). The cell suspension is then passed through a MACS column, which is placed in a magnetic separator. The CD14+monocytes are retained in the column, while the CD 14 fraction (which contains lymphocytes, including CD3+T cells) flows through. The column is then removed from the magnetic field, and the CD14+cells are eluted and resuspended for further use.

[0065] To isolate CD3+T cells from the CD 14 fraction, either MACS or FACS can be used. In MACS, the CD 14 fraction is incubated with anti-CD3 magnetic beads for 15 minutes at 4°C, followed by passage through a new MACScolumn. The CD3+cells are retained and later eluted for collection.

[0066] Alternatively, in FACS sorting, the CD 14 fraction is stained with fluorophore-conjugated anti-CD3 antibodies (such as CD3-PE or CD3-APC) and processed using a flow cytometer, where CD3+cells are gated and sorted. This method ensures the sequential isolation of total peripheral blood leukocytes, CD14+monocytes, and CD3+T cells, with high purity when properly optimized.

[0067] Detection of 4-HNE protein adducts

[0068] 4-HNE is a reactive a- B- aldehyde that contains three reactive groups: an aldehyde at Cl, a hydroxyl group at C4, and a double bond between C2 and C3. Hence, 4-HNE can form adducts with histidine, lysine, and cysteine residues of proteins and alter their functions (Dalleau et al., 2013; Grune & Davies, 2003). As used herein, the term ‘’4-HNE protein adducts” refers to proteins that are covalently modified by 4-HNE. Known methods to detect 4-HNE protein adducts include Western blotting, ELISA assays, or FACS analysis using anti-4-HNE antibodies and mass spectrometry (LC-MS, MALDI-TOF).

[0069] Preferably, in a method of the invention detection of 4-HNE protein adducts is performed using a specific binding partner directed against 4-HNE. As used herein, the term “binding partner directed against 4-HNE” refers to any molecule (natural or not) that is able to bind 4-HNE with high affinity. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably a monoclonal antibody. For example, the binding partner of 4-HNE of the invention is the mouse I IgGl monoclonal antibody Clone 12F7 against 4-HNE available from Invitrogen (MA5-27570).

[0070] The binding partner such as an 4-HNE antibody or 4-HNE aptamer may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (eitherdirectly or indirectly) a signal. Alternatively, the binding partner is detected indirectly, e.g., by using a secondary antibody labeled with a detectable molecule or substance. For example, a primary anti-4-HNE mouse I IgGl monoclonal antibody is suitably detected by a secondary (e.g., goat or donkey) anti-mouse IgH antibody, which is preferably conjugated to a fluorophore. Alternatively, the primary or secondary antibody can be conjugated to an enzyme, such as alkaline phosphatase or horseradish peroxidase (HRP), which converts a chromogen, or to luciferase for luminescence-based detection. Alternatively, the primary or secondary antibody can be conjugated to gold nanoparticles (AuNPs), magnetic beads, quantum dots, enzymes, colloidal particles, or other detectable moieties.

[0071] As used herein, the term "labeled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy 5)]) or radioactive molecule or a non-radioactive heavy metals isotopes or enzyme (e.g. HRP, alkaline phosphatase, luciferase) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance.

[0072] For 4-HNE protein adduct detection using in flow cytometry, the anti-4-HNE antibody may be either directly fluorophore-conjugated or it can be detected using a fluorophore-labeled secondary antibody.

[0073] Suitably, a method of the invention comprises the use of an unconjugated mouse monoclonal anti-4-HNE antibody, which requires a secondary antibody such as anti-mouse Alexa Fluor 488, PE, or APC for detection. In a preferred embodiment, a directly labeled (conjugated) anti-4-HNE antibody is used, like a FITC- or PE-conjugated version, which simplifies the protocolby eliminating the need for a secondary antibody. These antibodies are available from suppliers like Abeam, Millipore, R&D Systems, and Cayman Chemical.

[0074] To detect 4-HNE by flow cytometry, cells typically need to be fixed and permeabilized to allow antibody access to intracellular 4-HNE adducts. Fixation is typically performed with 4% PF A, followed by permeabilization with 0.1% Triton X-100 or a saponin -based buffer. After fixation, the cells are incubated with the primary anti-4-HNE antibody at an optimized dilution, usually ranging from 1:50 to 1:1000. If the primary antibody is unconjugated, a fluorophore-conjugated secondary antibody can be used for visualization. The stained cells are then analyzed by flow cytometry using an appropriate laser for the chosen fluorophore (e.g., 488 nm for FITC, 561 nm for PE).

[0075] In one embodiment, the method is performed using flow cytometry and comprises the steps of

[0076] - contacting an isolated sample from the subject with (fluorophore conjugated) antibodies against one or both of cell surface CD3 and CD 14 followed by fixating and permeabilizing the cell;

[0077] - staining the permeabilized cells with an antibody against 4-HNE adducts; - subjecting the stained sample to flow cytometry and gating for CD 14+ monocytes and / or CD3+T cells;

[0078] - determining the 4-HNE protein adduct level in CD14+ monocytes and / or CD3+T cells and, optionally, in total peripheral blood leukocytes.

[0079] The level of 4-HNE in a given cell population can be expressed as "Median Fluorescence Intensity” (MFI) which is used in the art as a relative measure of the amount of a given protein on or within a cell. However, it does not directly quantify the absolute number of protein molecules unless properlycalibrated. MFI is often used to compare protein expression between different conditions (e.g., treated vs. untreated cells or diseased cells vs. healthy cells) but does not provide an absolute count of molecules per cell. If absolute quantification is needed, MFI can be converted into actual protein or receptor numbers using quantitative flow cytometry techniques, such as bead-based calibration (e.g., MESF or ABC beads) using beads having known amounts of fluorophore or antibody-binding capacity and allow for calibration. Fluorescence-tagged antibodies with known binding efficiency can be used to estimate protein copies per cell. Factors like fluorophore brightness, antibody binding efficiency, instrument settings, and autofluorescence can affect MFI, so it is preferred to include controls for making accurate comparisons.

[0080] A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.

[0081] "Providing a diagnosis” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a sepsis condition or to one at risk of developing a sepsis. It may also comprise riskevaluation, such as a prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of sepsis, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population.

[0082] A method according to the invention may further comprise the use of a clinical scoring approach. In one aspect, it comprises subjecting a subject to a standardized system used to assess and monitor acutely ill patients, such as the National Early Warning Score (NEWS) scoring. NEWS helps healthcare providers detect early signs of clinical deterioration and intervene promptly. The scoring is based on six physiological parameters: respiratory rate, oxygen saturation, systolic blood pressure, pulse rate, level of consciousness (using the AVPU scale), and temperature. An additional point is added if the patient requires supplemental oxygen. Each parameter is assigned a score from 0 to 3, depending on how abnormal the readings are. The total NEWS score determines the level of clinical concern. A score of 0 to 4 indicates low risk and routine monitoring is sufficient. A score of 5 or 6 suggests medium risk, requiring urgent assessment and possible escalation. A score of 7 or more indicates high risk, requiring an emergency response and possible transfer to critical care. As disclosed herein below, it was found that stratifying the patients based on reduction in 4-HNE adduct levels in CD3 / CD14 cell populations could improve the diagnostic performance of NEWS indexes below of equal to 8. For example, the NEWS scoring showed an AUG of 92.0% for the about 20% patients with the lowest levels of 4-HNE protein adduct levels in CD3 lymphocytes, compared to 82.7% without stratification based on 4-HNE. Reduced 4-HNE protein adduct levels in CD3+T cells, and ratios of 4-HNE protein adduct levels in CD3+T cells and / or CD 14+ monocytes over total leukocyte adduct levels also improve the diagnostic performance of patients that are first stratified byNEWS. In one embodiment, a method of the invention comprises selecting a subject having a NEWS scoring < 8, preferably <6, more preferably <5. Also provided herein is the use of detecting 4-HNE protein adduct levels in CD3+T cells and / or CD 14+ monocytes to improve the diagnostic outcome of NEWS for predicting sepsis severe outcome.

[0083] A method of the invention suitably comprises detection of one or more factors or biomarkers implicated in sepsis. The factor(s) or biomarker(s) may have been previously implicated in (early) sepsis. In one aspect, it comprises detecting an analyte which is related or linked to the functional metabolic and / or transcriptional consequence of 4-HNE protein modulation in immune cells during sepsis. This may include cytokine secretion (e.g., IL- 10, IL-6), DNA damage, and transcriptional regulation. In one embodiment, a method comprises determining the level of one or more cytokines, preferably selected from the group consisting of IL- 10, IL-6 and TNF-a.

[0084] In one embodiment, a method further comprises selecting the subject for treatment with an anti-sepsis treatment. Another object of the present invention is a method of treating sepsis in a subject comprising selecting a subject that suffers from (early) sepsis or is at risk of developing sepsis by a method as described herein above, followed by treating the subject with an anti-sepsis treatment. Preferably, said anti-sepsis treatment comprises one or more of antibiotics, vasopressors and the use of intravenous fluids.

[0085] In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.Examples of antibiotic agents include, without limitation, penicillin, quinoline, vancomycin, sulfonamides, ampicillin, ciprofloxacin, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, chloramphenicol and sulfisoxazole.

[0086] Diagnostic Device for Detection of Sepsis

[0087] In a further aspect, the present invention provides a diagnostic device designed to detect biomarkers associated with sepsis, incorporating the means for carrying out parts of the method described earlier in this application. Preferably, the diagnostic device is configured as a point-of-care (PoC) test device, making it suitable for rapid and efficient use in clinical environments, such as hospitals, emergency rooms, or physician’s offices, where immediate diagnostic results are required.

[0088] The diagnostic device (1) may comprise an elongated housing (2) that defines the overall structure and form of the device. This housing may comprise several components, such as a fluid sampling chamber, and a viewing window, ensuring ease of handling and operation in a clinical setting. The housing can be made from durable materials and is suitably designed for portability, facilitating use in fast-paced, point-of-care environments.

[0089] A viewing window is integrated into the elongated housing, providing the user with clear visibility of the results as they appear on the lateral flow test strip. The results are typically observed as color changes at the test lines, which are indicative of the presence or absence of specific biomarkers associated with sepsis. The viewing window enables the user to easily read and interpret the results, providing quick feedback for clinical decisionmaking.

[0090] A fluid sampling chamber serves as the entry point for the biological sample, typically a blood sample, to be introduced into the device. Thischamber is connected to the test strip holder, ensuring proper migration of the sample onto the lateral flow test strip. The device may further incorporate a test strip holder that contains a lateral flow test strip. This test strip is equipped with means for detecting 4-HNE protein adducts, for example, a specific binding partner such as an antibody or aptamer that specifically binds to 4-HNE. The 4-HNE binding partner is preferably conjugated to a detectable label or particle, such as gold nanoparticles or other detectable tags, enabling the visual or instrumental detection and quantification of 4-HNE protein adducts.

[0091] Alternatively, a labeled secondary detection probe, which complexes with the 4-HNE binding partner, can be used to detect 4-HNE protein adducts in the respective cell populations. This secondary probe, such as an antibody, may carry a detectable label, such as fluorochromes, enzymatic markers, or colloidal gold, which facilitates the reading of the assay results.

[0092] Specifically, the test strip may contain a conjugation zone, wherein antibodies or aptamers against 4-HNE are dispersed within the lateral flow test strip. The conjugation zone serves as the region where the sample, once introduced, mixes with the labeled binding agents. Upon interaction with 4-HNE protein adducts in the sample, the 4-HNE binding partner forms complexes, which migrate along the test strip. These complexes are then detected at the capture zone or test zone with immobilized e.g. in a line form of cell population-specific "capturing” antibodies, such as anti-CD14 for monocytes or anti-CD3 for T cells. The accumulation of the labeled complexes at these capture zones generates a detectable signal, confirming the presence of 4-HNE protein adducts in the captured immune cells.

[0093] This configuration allows for both the detection of 4-HNE protein adducts and the selective capture of specific immune cell populations, making thedevice particularly useful for diagnosing and monitoring sepsis or other inflammatory conditions where oxidative stress is implicated.

[0094] The test strip may also feature one or more further capture zone(s), designed to capture different alternative cell populations, e.g. leukocytes or neutrophils. For example, one capture zone serving as "test zone” may contain immobilized anti-CD3 antibodies which specifically capture T cells, while another capture zone, serving as "control zone” may contain anti-CD45 antibodies, which capture all leukocytes, and / or anti-CD15 antibodies, which capture neutrophils, to confirm that the test is performed correctly. See for example Figure 10. Once the sample has migrated through the test strip, the detection of 4-HNE protein adducts in the respective cell populations (capture zones) provides valuable diagnostic information. This design allows for a more comprehensive analysis of the immune response.

[0095] In addition to detecting 4-HNE protein adducts in T cells and monocytes, the lateral flow test strip may be further configured to detect additional biomarkers relevant to sepsis and the immune response, such as cytokines. These cytokines, including interleukins (such as IL-6 and IL- 10) and TNF-a, are elevated in the inflammatory response seen during sepsis and can provide important diagnostic insights. The test strip may be equipped with additional capture zones specifically designed to detect these cytokines, adding another layer of diagnostic capability to the device.

[0096] The diagnostic device usually operates by first receiving the biological sample, which is typically blood, and processing it through the fluid sampling chamber. Once the sample is applied, it migrates along the lateral flow test strip e.g. by capillary action through a porous membrane such as a nitrocellulose membrane.As the sample passes through the conjugate region, target analytes (if present) form complexes with the labeled antibodies. The resulting complexes continue to migrate along the membrane to at least one capture zone (test line), comprising immobilized binding agents specific for cell-type-associated markers, such as CD3 and / or CD 14, thereby capturing complexes associated with T cells and / or monocytes. The presence of 4-HNE protein adducts in the captured T cells and monocytes generates a detectable signal, visible as a color change at the test line.

[0097] In one embodiment, the device comprises:

[0098] an elongate housing defining a test strip holder containing a lateral flow test strip comprising, preferably immobilized, means for detecting 4-HNE protein adducts, and antibodies against one or both of CD3 and CD 14, a fluid sampling chamber provided with an opening connecting the fluid sampling chamber with the test strip holder, and

[0099] a viewing window in the elongate housing allowing reading of one or more portions of the lateral flow test strip.

[0100] For example, the lateral flow test strip comprises an (immobilized) antibody or aptamer against 4-HNE protein, and antibodies against CD3 and / or CD 14 to isolate the cell population(s) of interest. For example, the test strip comprises a first “collection” band with anti-CD3 antibody (for capturing T cells) and a second “collection” band with anti-CD45 antibody (for capturing all leukocytes). This strip may then be probed with a detection probe linked to a 4-HNE antibody in order to detect 4-HNE protein adducts in the respective cell populations.

[0101] In some embodiments, the device also includes a control zone that ensures the proper functioning of the assay. This control zone contains immobilized binding agents specific to an alternative cell marker, such as CD 15, which captures complexes associated with neutrophils, or an anti-CD45 antibodyfor capturing all leukocytes. The presence of a signal at the control line confirms that the test has worked correctly and that the sample has migrated properly through the test strip.

[0102] The results of the assay are suitably interpreted by comparing the intensity of the signal at the test line (for T cells and monocytes) with the signal at the control line (for neutrophils). A relative decrease in signal intensity at the test line, compared to the control line, is indicative of sepsis. This suggests a disruption in the immune system, as seen in sepsis, where certain immune cell populations, like T cells and monocytes, may be altered.

[0103] This diagnostic device can also be broadened to detect a wide range of biomarkers associated with other diseases and inflammatory conditions. For example, the device can be adapted to detect different forms of oxidative stress or cytokine profiles relevant to other disorders. In one aspect, a flow test strip may further comprise means for detecting additional markers of relevance for sepsis, such as cytokine(s), like interleukins (e.g. IL-6, IL- 10) and / or TNF-a. The test strip may be multiplexed to detect multiple biomarkers simultaneously, enhancing diagnostic accuracy and providing a broader view of the immune response. Moreover, the device can be used with other biological fluids, such as urine, saliva, or cerebrospinal fluid, broadening its applications for different clinical scenarios.

[0104] Also provided is a kit-of-parts comprising (i) means for detecting 4-HNE protein adducts and (ii) means for capturing CD 14+ monocytes and / or CD3+ T cells. The components (i) and (ii) may be provided in separate containers. Suitably, the kit comprises aptamers, antibodies or antigen-binding fragments thereof capable of binding 4-HNE. In one aspect, the means for detecting 4-HNE protein adducts are conjugated to a detectable label, preferably selected from fluorochromes, gold nanoparticles (AuNPs), magnetic beads, quantum dots, enzymes, colloidal particles, or other detectable moieties. In some embodiments, the means for capturing CD 14+monocytes and / or CD3+ T cells are antibodies or antigen-binding fragments thereof. The kit may comprise one or more of: (a) a sample collection device; (b) a reaction vessel or test strip; (c) a buffer or reagent solution; (d) a detection device or reader; and / or (e) instructions for use. The kit may further comprise instructions for use in detecting 4-HNE protein adducts in CD 14+ monocytes and / or CD3+ T cells as a diagnostic indicator for sepsis, preferably early-stage sepsis.

[0105] LEGEND TO THE FIGURES

[0106] Figure 1: Gating strategy of buffy coat from a healthy individual treated with 10 pM or 50 pM 4-HNE. (A) Identification of cells based on forward scatter-height (FSC-H) and side scatter-height (SSC-H). (B) selection of single cells based on FSC-H and area FSC-A. (C) determination of 4-HNE positive cells based on labeling with a FITC-conjugated antibody against 4-HNE. (D) Histograms showing 4-HNE signal (mode: normalized to maximum) for unstained cells, untreated cells stained with anti-4-HNE-FITC, and cells treated with 10 or 50 pM 4-HNE stained with anti-4-HNE-FITC.

[0107] Figure 2: Total 4-HNE protein adduct levels in patient control, sepsis non-severe, and sepsis severe outcomes (i.e., ICU admission, death, or a rise in SOFA-score of two or more within 72 h). Buffy coats were stained with antibodies against 4-HNE protein adducts, CD3 and CD 14, and analyzed by flow cytometry. Panel A indicates the level of total 4-HNE protein adducts expressed as Median Fluorescence Intensity (MFI). Histograms for representative patients of each group are shown in Panel B.

[0108] Figure 3: Gating strategy of Buffy coats stained with CD3+, CD14+, and 4-HNE. Buffy coats of sepsis patients were stained for different cell surfacemarkers and intracellular 4-HNE protein adducts. The gating strategy displayed is used for flow cytometry analysis. First, cells were identified based on forward scatter-area (FSC-A) and side scatter-area (SSC-A). See panel A. Second, single cells were selected based on forward scatter-height (FSC-H) and area FSC-A. See panel B. Third, CD3+ T cells and CD 14+ monocytes were identified based on labeling with fluorescently labeled antibodies, and 4-HNE positive cells were identified based on labeling with a FITC-conjugated antibody against 4-HNE. See panel C.

[0109] Figure 4: 4-HNE protein adduct levels in CD 14+ monocytes. Panel A indicates the ratio of 4-HNE protein adducts on CD 14+ monocytes (expressed as Median Fluorescence Intensity (MFI)) over total peripheral blood leukocytes in control subjects without sepsis, sepsis patients with non-severe outcome and sepsis patients with severe outcome. Histograms for representative patients of each group are shown in Panel B.

[0110] Figure 5: 4-HNE protein adduct levels in CD3+T cells. Panel A indicates the ratio of 4-HNE protein adducts on CD3+ T cells (expressed as Median Fluorescence Intensity (MFI)) over total peripheral blood leukocytes in control subjects without sepsis, sepsis patients with non-severe outcome and sepsis patients with severe outcome. Histograms for representative patients of each group are shown in Panel B.

[0111] Figure 6: Receiver operating characteristic (ROC) curve analysis for diagnostic performance of 4-HNE protein adducts for sepsis compared to non-septic control patients. (A) Total 4-HNE protein adduct levels in huffy coat leukocytes (4-HNE increased in sepsis). (B) CD3+T cells (4-HNE decreased in sepsis). (C) The ratio of CD3+T cells over leukocytes (decreased ratio). The ROC analyzes the ability of 4-HNE to discriminate sepsis patients from age-matched non-sepsis control patients. The areaunder the ROC curves (AUG) are: panel A: 57.6%; panel B: 74.8%; panel C: 66.0%. Note that the reduction of 4-HNE in CD3+cells and the ratio of 4-HNE in CD3 / leukocytes have a substantially larger diagnostic performance than 4-HNE alone.

[0112] Figure 7: Receiver operating characteristic (ROC) curve analysis for recognizing sepsis patients that will develop severe outcome (admission to the ICU and death within 72 hours of admission) based on NEWS (National Early Warning Score), with and without patient stratification based on 4-HNE protein adduct levels. (A) Comparison of NEWS for patients stratified by low levels of 4-HNE protein adducts on CD3+T lymphocytes. AUG without stratification (all patients): 82.3%. AUG with stratification (lowest 20% patients): 93.8%. (B) Comparison of NEWS for patients stratified by low ratios of 4-HNE protein adducts on CD3+T lymphocytes over total 4-HNE protein adducts. AUG without stratification (all patients): 82.3%. AUG with stratification (-50% patients): 87.4%.

[0113] Figure 8: 4-HNE protein adduct levels and ratios improve the diagnostic performance of NEWS for predicting sepsis severe outcome (admission to the ICU and death within 72 hours of admission). The x-axis shows the NEWS (National Early Warning Score) score and the y-axis shows Youden’s J statistic (calculated as the difference between the true positives (sensitivity) and true negatives (specificity); a higher value indicates better diagnostic performance). As apparent from the graph, for NEWS indexes below or equal to 8, 4-HNE protein adducts levels improve the diagnostic performance. Moreover, reduced 4-HNE protein adduct levels on CD3+ T cells and / or CD 14+ monocytes perform substantially better for sepsis outcome diagnosis than the increase in total leukocyte 4-HNE protein adduct levels.Figure 9. Total levels of 4-HNE protein adducts in CD 15+ CD66b+neutrophils isolated from peripheral blood of sepsis patients (n = 5;

[0114] "Sepsis”), patients with uncomplicated infection (n = 7; "Infection”) or healthy controls (n = 5, "no infection”). Neutrophils were isolated by magnetic separation and stained with antibodies specific for 4-HNE protein adducts, CD 15, and CD66b, followed by flow cytometric analysis. The y-axis represents 4-HNE protein adduct levels, expressed as median fluorescence intensity (MFI).

[0115] Figure 10. Exemplary colloidal gold lateral flow immunoassay (LFIA) device for detecting sepsis, the device (1) comprising a test strip holder (2) containing a lateral flow test strip; a fluid sampling chamber (3); a conjugation zone (4) comprising dispersed therein means for detecting 4-HNE protein adducts; a capturing zone (5) comprising immobilized antibodies against one or both of CD3 and CD 14; and a control zone (6) comprising immobilized antibodies against CD 15.

[0116] EXPERIMENTAL SECTION

[0117] In the examples 1-3 below, the role of 4-HNE in sepsis is addressed. First, we investigated the levels of 4-HNE protein adducts in CD 14+ monocytes and CD3+T cells in huffy coats obtained from patients at the emergency department with either sepsis with a severe outcome (i.e., ICU admission, death, or a rise in SOFA-score of two or more within 72 h), sepsis without a severe outcome, and a control group without sepsis. These 4-HNE adduct levels were correlated with plasma cytokine levels.EXAMPLE 1: Clinical study design and obtaining patient blood samples.

[0118] To determine the 4-HNE protein adduct levels, huffy coats and serum samples were selected from the Acutelines biobank from three groups of patients (Table 1). Acutelines is a data biobank at the Emergency Department (ED) of the University Medical Center Groningen (UMCG) that prospectively collects data and biomaterials from acutely ill patients. The ED nurse and a researcher screen patients for eligibility upon arrival at the ED. Upon triage, before initiation of treatment, blood samples were collected in EDTA tubes, centrifuged for 15 min at 1,500 x g, and stored at -80°C in the Acutelines biobank. Bedside data monitored (i.e., vital parameters) are automatically captured and stored, and information from other data sources, such as the electronic health records of the hospital, emergency medical services-, and the general practitioner, the municipal registration, health insurance companies, and the pharmacy, is securely imported.

[0119] The patient groups were selected to ensure comparability in gender and age distribution. Patients in sepsis groups have confirmed bacterial infection, for 1 / 3 of the patients in both severe and non-severe outcomes accompanied by a viral infection. Sepsis severe outcome is defined as patients diagnosed with sepsis who were admitted to the ICU and died within 72 hours of admission.

[0120] A compensated vasodilated state is commonly observed in sepsis patients, often resulting in a significant decrease in diastolic blood pressure (DBP) while systolic blood pressure (SBP) remains stable. This state can act as a warning sign of impending cardiovascular collapse (Holder et al., 2016). In this study, detailed clinical assessments showed that DBP was notably lower in the sepsis group compared to the control group, while SBP remained unchanged. Moreover, the body temperature in sepsis with non-severe and severe outcomes was significantly increased. Septic patients withsevere outcomes displayed a higher decrease in consciousness compared to septic patients without severe outcomes.

[0121] The Sequential Organ Failure Assessment (SOFA) score is a widely used tool to evaluate the severity of organ dysfunction in critically ill patients, particularly those with sepsis. It quantifies the degree of dysfunction across multiple organ systems, with higher scores indicating more severe organ failure and an increased mortality risk (Boekhoud et al., 2024). In this study, it was observed that both sepsis non-severe and sepsis severe outcomes initially had a SOFA score higher than 2. However, in non-severe cases, the score was reduced to 0, while in severe cases, it remained above 2 even 72 hours after admission to the ED.

[0122] The hematological parameters (Table 2) show that C-reactive protein (GRP), IL-6, and IL- 10 increased significantly in both sepsis groups, with the highest increase in the severe outcome group.Table 1: Patient characteristics and clinical outcomes*

[0123] Parameter Control Sepsis Sepsis + severe outcome Age (years) 70 (62-77.25) 64.5 (63± 14.4) 70 (60.5-73.5) Male sex (n [%]) 22 (61%) 19 (55.9%) 21 (60%) Infection type

[0124] Bacterial (n [%]) 0 34 (97%) 35 (100%) Viral (n [%]) 0 10 (28.57%) 9 (25.71%) Vital parameters at

[0125] triage 100 (97.97 ± 17.43) 99.5 (88.5-109.5) 103 (107.29 ± 22.69) Heart rate (bpm) 134 (137.2 ± 26.61) 121 (124.61 ± 23.45) 120 (121.7353 ± SBP pressure 85 (84.28 ± 15.55) 69 (62-81.5)* 28.84)* (mmHg) 20 (19.9 ± 4.51) 20 (18-24) 76 (77.24 ± 15.11) DBP (mmHg) 36.5 (36.6 ± 0.81) 37.5 (36.5-38.3)* 24.5 (25.28 ± 7.50)* Respiratory rate 34 (94.44%) 32 (94.12%) 37 (37.30 ± 1.20) ( / min) 1 (2.78%) 1 (2.94%) 17 (48.57%) Body temperature 0 (0%) 0 (0%) 8 (22.86%) (°C) 10 (28.57%) EMV score 15 (n [%])

[0126] EMV score 13-14 (n

[0127] [%])

[0128] EMV score < 12 (n

[0129] [%])

[0130] SOFA score

[0131] 24 hours 2 (1-2.25) 2.5 (2-1.4)* 7 (6.47 ± 2.97)* 48 hours 0 (0-1) 1 (0-1.75) 4 (1-7)* 72 hours 0 (0-0) 0 (0-1) 3 (0-5.25)* ICU admission < 72 h 0 0 25 (71.42%)

[0132]

[0133] Death < 72 h 0 0 9 (25.71%)

[0134] *SBP: systolic blood pressure, DBP: diastolic blood pressure, EMV: Eye-Motor-Verbal (Glasgow coma score), SOFA: sequential organ failure assessment, ICU: intensive care unit. Data are presented as median (mean ± SD) when normally distributed, as median (IQR25-IQR75) when not normally distributed, or (relative) number as indicated. Shapiro-Wilk test was used to test for normal distribution of data. Not normally distributed data significance was tested with the Kruskal-Wallis test , otherwise with ONE-WAY ANOVA. Everything was compared to the control group. * indicates p*<0.05.Table 2: Hematological and biochemical parameters upon triage at the ED* Parameter Control Sepsis Sepsis + severe outcome Leukocyte count (x 10.4 (9.83 ± 4.62) 12.55 (8.65-16.05) 12.5 (12.98 ± 7.97) 109 / L)

[0135] Plasma bilirubin 8.5 (8.19 ± 3.29) 11 (6.5-15.5) 10.5 (6.25-21.5) (pmol / L)

[0136] Plasma creatinine 94 (71.75-136.5) 98 (66.25-183.75) 122 (84-217) (pmol / L)

[0137] Plasma GRP 14.5 (3.7-33.5) 129.5 (52.25- 67 (32.5-187.5)* (mg / dL) 202.25)*

[0138] Serum IL-6 (pg / mL) 48.35 (41.40- 125.72 (70.13- 253.86 (84.8- 63.57) 321.42)* 2037.05)* Serum IL- 10 22.77 (14.69- 41.53 (21.35-68.57)* 74.65 (35.95- (pg / mL)) 37.11) 146.71)* Serum TNF -a 392.68 (240.05- 312.97 (226.34- 291.49 (230.16- (pg / mL) 481.26) 439.92) 419.45)

[0139]

[0140] *Data are presented as median (mean ± SD) when normally distributed, as median (IQR25-IQR75) when not normally distributed, or (relative) number as indicated. Shapiro-Wilk test was used to test for normal distribution of data. Normally distributed data significance was tested with the Kruskal-Wallis test, otherwise with ONE-WAY ANOVA. Everything was compared to the control group. * indicates p*<0.05.EXAMPLE 2: 4-HNE adducts are increased in patients with sepsis but decreased in monocytes and T cells.

[0141] For measuring 4-HNE protein adduct levels, the detection method was first tested on a huffy coat isolated from a healthy donor that was frozen immediately after drawing blood. This procedure mimics the huffy coat collection procedure of the Acutelines biobank. The huffy coat from the healthy donor was treated with 10 pM or 50 pM of 4-HNE for 30 min before being stained with an antibody against 4-HNE-modified proteins.

[0142] The huffy coat was used to detect 4-HNE protein adducts in total cells, CD 14+ monocytes, and CD3+ T cells. The huffy coats were thawed and washed several times in cold PBS. The cells were stained with anti-human CD3 antibody (Miltenyi, REAfinity 130-113-141, 1:50) and CD14 antibody (Miltenyi, REAfinity 130-110-583, 1:50) for 10 min at 4°C. Subsequently, cells were washed with cold PBS before fixation with 4% paraformaldehyde (PF A) (Aurion, 15710) for 15 min at 4°C. After fixation, the cells were washed several times in cold PBS, followed by blocking and permeabilization in PBS containing 2% human serum (Sigma / MERCK, H4522-20ML) and 0.05% saponin (Sigma, 47036-50G) for 30 min at 4°C. Subsequently, permeabilized cells were incubated for 1 hour with primary antibody for 4-HNE protein adducts (Invitrogen, MA5-27570, 1:60) in 2% human serum (Sigma / MERCK, H4522-20ML) and 0.05% saponin at 4°C. Cells were washed three times again with PBS containing 2% human serum and 0.05% saponin before being incubated with secondary antibody donkey-anti mouse IgG (H&L) Alexa flour 488 (ThermoScientific, 10544773, 1:400) for 30 min at 4°C.

[0143] Finally, the cells were washed twice in PBS before analyzing the samples using a CytoFlex S benchtop flow cytometer (Beckman Coulter). Data were analyzed using NovoExpress Software (Agilent). Figure 1A-C summarizes the gating strategy for the flow cytometry. As shown in FigureID, the level of 4-HNE protein adducts increased with an increasing dose of 4-HNE as measured by flow cytometry.

[0144] Next, we analyzed 4-HNE protein adducts in huffy coats obtained from patients. Patients with sepsis without- or with a severe outcome (i.e., ICU admission or death < 72 hours) show, on average, 7-fold increased levels of 4-HNE protein adducts in the buffy coat as compared to acutely ill patients without an infection that served as the control group (Figure 2). These results are in line with the increase in malondialdehyde (MDA), which is another lipid peroxidation product, observed previously in the plasma of sepsis patients (van der Slikke et al., 2022).

[0145] Since the flow cytometry approach allows for discerning 4-HNE protein adduct levels in different immune cell types, cells were co-stained for CD 14 and CD3 to determine 4-HNE protein adduct levels in CD 14+ monocytes and CD3+T cells, respectively. The gating strategy is shown in Figure 3.

[0146] Surprisingly, the levels of 4-HNE protein adducts in the CD14+ (Figure 4) and CD3+(Figure 5) cells were significantly lower in the sepsis patient groups compared to the healthy donors. For CD 14+ cells, the ratios were on average >2-fold lower in the sepsis patient groups compared to the healthy donors. For CD3+cells, the ratios were on average >9-fold lower in the sepsis patient groups compared to the healthy donors. Thus, the increased 4-HNE levels, as observed in buffy coat data, are mostly contributed to by the CD 14’ CD3_population, which most likely comprises neutrophils (CD 15+) and neutral killer (NK) cells (CD3-, CD56+).

[0147] EXAMPLE 3: Diagnostic performance of the novel sepsis assay.

[0148] Whereas 4-HNE protein adduct levels are increased in sepsis patients compared to control patients, this has only low diagnostic performance, withan area under the curve (AUG) of only 57.6% in the receiver operating characteristic (ROC) curve (Figure 6A). In contrast, our novel data show that 4-HNE protein adduct levels are reduced in CD3+T cells of sepsis patients and, moreover, that this reduction has a substantially better diagnostic performance with an AUG of 74.8% (Figure 6B).

[0149] A disadvantage of using absolute 4-HNE protein adduct levels is that a diagnostic test based on this parameter will require calibration and / or an external standard for comparison. This problem can be overcome by using the ratio of 4-HNE protein adducts on T cells and / or CD 14+ monocytes (both decreased in sepsis) over the total 4-HNE protein adduct levels (increased in sepsis) in a blood sample. Herewith, an external standard is no longer required. This ratio has a diagnostic performance of 66% AUG for CD3+lymphocytes (Figure 6C).

[0150] In addition to identifying patients with sepsis, early recognition of those at risk of developing severe sepsis outcomes, as opposed to those likely to have milder cases, allows for more targeted and effective treatment. The severity of sepsis is currently predicted using NEWS (National Early Warning Score) scoring. NEWS is a tool used in medical settings to assess and monitor a patient's condition, particularly for identifying early signs of clinical deterioration, such as sepsis. It helps to standardize the detection of acute illnesses and improve patient outcomes. It is based on 6 clinical parameters that can be readily assessed (respiratory rate, oxygen saturation, systolic blood pressure, heart rate, level of consciousness, temperature). These values are given a scoring, with a score >5 indicating a higher risk of deterioration. However, in a systematic review of studies of patients with infection receiving care outside an intensive care unit, a NEWS score >5 predicted death with an AUG of only 80% [Zhang K, Zhang X, Ding W, et al. National Early Warning Score does not accurately predict mortality for patients with infection outside the intensive care unit: a systematic reviewand met a- analysis. Front Med 2021;8:704358], In our study, NEWS was able to predict severe outcome, defined as patients diagnosed with sepsis who were admitted to the ICU and died within 72 hours of admission, with a similar performance as published, as we obtained an AUG of 82.7% (Figure 7).

[0151] Therefore, it was assessed whether 4-HNE protein adduct levels can improve the diagnostic performance of NEWS. Our data surprisingly show that stratifying the patients based on 4-HNE protein adduct levels in CD3+lymphocytes substantially improves NEWS (See Table 3).

[0152] Specifically, the NEWS scoring showed an AUG of 92.0% for the about 20% patients with the lowest levels of 4-HNE protein adduct levels in CD3+lymphocytes, compared to 82.7% without stratification based on 4-HNE (Figure 7 A). Stratification based on the ratio of 4-HNE protein adducts on T cells over the total 4-HNE protein adduct levels also improved the AUG of NEWS to 87.6% (Figure 7B). In contrast, stratification based on total 4-HNE protein adduct levels did not improve the AUG of NEWS for sepsis outcome.

[0153] Moreover, 4-HNE protein adduct levels on CD3+ T cells, and ratios of 4-HNE protein adduct levels on CD3+ T cells and / or CD 14+ monocytes over total leukocyte adduct levels also improve the diagnostic performance of patients that are first stratified by NEWS, as indicated by the improved Youden’s J statistic for NEWS scores below 8 (Figure 8).Table 3. Diagnostic performance of NEWS for outcome prediction of sepsis.

[0154] Stratification Ground truth Sensitivity1Specificity1Positive Negative %

[0155] 72 ICU Survival (% true (% true predictive predictive patients admission and / or positive) negatives) value1value1in group or discharge

[0156] mortality

[0157] All patients 35 69 80.0% 55.1% 47.5% 84.4% 100% Low levels of 8 11 87.5% 54.5% 58.3% 85.7% 18.3% 4-HNE protein

[0158] adducts in CD3

[0159] lymphocytes

[0160] Low ratio of 4- 18 31 83.3% 61.3 55.6% 86.4% 47.1% HNE protein

[0161] adducts in CD3

[0162] compared to

[0163]

[0164] total

[0165] 'Calculated for NEWS score >5, the clinically used score for identification of sepsis.

[0166] EXAMPLE 4: 4-HNE Adduct Levels in Neutrophils of Sepsis Patients

[0167] As demonstrated in Example 2, the levels of 4-hydroxynonenal (4-HNE) adducts are generally elevated in patients with sepsis (Figure 2), while a reduction in 4-HNE adduct levels was observed specifically in CD 14+ monocytes (Figure 4) and CD3+T cells (Figure 5). This example highlights that the decrease in 4-HNE adduct levels is cell-type specific, with neutrophils — representing the most abundant white blood cell population in peripheral blood (approximately 50-70% of total leukocytes) — not exhibiting the same reduction seen in other cell types.

[0168] Blood Sample Collection and Neutrophil Isolation

[0169] Blood samples were collected from peripheral blood of three distinct patient groups: healthy controls (n = 5), sepsis patients (n = 5), and patients with uncomplicated infections (n = 7). Neutrophils were isolated from whole blood using the MACSXpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec) in accordance with the manufacturer's instructions. Briefly, 1 mL of whole blood was mixed with the isolation buffer and gently inverted for 5 minutes at room temperature (RT). The sample was then processed using the MACSXpress Separator and incubated for 15 minutes at RT. Thesupernatant containing plasma and neutrophils was carefully collected, and the neutrophil concentration was determined using the Scepter 3.0 Cell Counter (Merck Millipore).

[0170] Flow Cytometry for 4-HNE Adduct Detection

[0171] Purified neutrophils (2 x 105cells) were incubated in flow cytometry buffer (PBS + 2 mM EDTA + 0.5% BSA) containing fluorochrome-conjugated antibodies against CD 15 (PE) and CD66b (VioBright V600) for extracellular staining. This incubation was performed for 30 minutes at 4°C in the dark. Following extracellular staining, cells were fixed and permeabilized using the Inside Stain Kit (Miltenyi Biotec). The cells were first incubated with Inside FIX for 20 minutes at RT, followed by a wash. Permeabilization was then performed in Inside PERM supplemented with 2% human serum for 30 minutes at 4°C in the dark.

[0172] Subsequently, cells were incubated with a primary antibody against 4-hydroxynonenal (4-HNE, Invitrogen) Subsequently, cells were incubated with a primary antibody against 4-hydroxynonenal (4-HNE, Invitrogen) for 30 minutes at 4°C, followed by incubation with a donkey anti-mouse Alexa Fluor 488 secondary antibody (Thermo Fisher) for 30 minutes at 4°C in the dark. After a final wash, the cells were resuspended in flow cytometry buffer and analyzed using the NovoCyte Quanteon (Agilent Technologies).

[0173] Neutrophils were gated as CD15+CD66b+cells within the population of single cells.

[0174] Results

[0175] The data presented in Figure 9 demonstrate that 4-HNE adduct levels in neutrophils from sepsis patients are either elevated or comparable to those observed in neutrophils from healthy controls and patients with uncomplicated infections.These results demonstrate that the reduction in 4-HNE adduct levels observed in monocytes and T cells during sepsis is not a universal response across all immune cell types. Specifically, neutrophils, as the predominant white blood cell population in peripheral blood, do not show a decrease in 4-HNE adducts, underscoring the cell-type specificity of a reduction in 4-HNE adduct levels during sepsis.

[0176] EXAMPLE 5: Lateral flow immunoassay for detection of sepsis-associated biomarkers.

[0177] This Example describes an exemplary lateral flow immunoassay (LFIA) and its use for the detection of sepsis-associated alterations in 4-HNE protein adduct levels within specific leukocyte populations. A schematic of the exemplary LFIA is shown in Figure 10.

[0178] A blood sample is obtained from a subject. Red blood cells are lysed to enrich the sample for leukocytes. The processed sample is then applied to the fluid sampling chamber of a diagnostic device comprising an elongated housing containing a test strip holder with a lateral flow test strip.

[0179] The lateral flow test strip is configured with labeled binding agents specific for 4-HNE protein adducts, e.g., antibodies against 4-HNE which are conjugated to detectable particles such as colloidal gold. Upon application of the sample, it migrates along a porous membrane, such as nitrocellulose, by capillary action. As the sample passes through the conjugate region of the test strip, target analytes will form complexes with the labeled binding agents. These complexes then continue to migrate along the membrane toward the capture zone(s).The capture zones (or test lines) comprise immobilized binding agents specific for cell-type-associated markers, such as CD3 and / or CD 14, which are markers for T cells and monocytes, respectively. These binding agents capture the complexes associated with T cells and monocytes. The accumulation of labeled complexes at the test line generates a detectable signal, which can be observed via the viewing window in the elongated housing.

[0180] The device preferably also comprises a control zone (or control line) containing immobilized binding agents specific for an alternative cell population marker, such as CD 15, which is a marker for neutrophils. This control zone captures labeled complexes associated with neutrophils and ensures proper assay function.

[0181] Sepsis is determined by comparing the intensity of the signal at the test line with the intensity of the signal at the control line. A relative decrease in the signal at the test line (indicating reduced binding of complexes to T cells and / or monocytes), compared to the control line (which is associated with neutrophils), is indicative of sepsis.

[0182] REFERENCES

[0183] Boekhoud, L. et al. Predictive performance of NEWS and qSOFAin immunocompromised sepsis patients at the emergency department.

[0184] Infection 52, 1863-1873 (2024).

[0185] Brealey, D. et al. Association between mitochondrial dysfunction and severity and outcome of septic shock. The Lancet 360, 219-223 (2002).

[0186] Dalleau, S., Baradat, M., Gueraud, F. & Hue, L. Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance. Cell Death Differ 20, 1615-1630 (2013).Dwivedi, S., Sharma, A., Patrick, B., Sharma, R. & Awasthi, Y. C. Role of 4-hydroxynonenal and its metabolites in signaling. Redox Report 12, 4-10 (2007).

[0187] Galley, H. F. Oxidative stress and mitochondrial dysfunction in sepsis. Br J Anaesth 107, 57-64 (2011).

[0188] Grune, T. & Davies, K. J. A. The proteasomal system and HNE-modified proteins. Mol Aspects Med 24, 195-204 (2003).

[0189] Holder, A. L. et al. Predictors of early progression to severe sepsis or shock among emergency department patients with nonsevere sepsis. Int J EmergMed 9, 10 (2016).

[0190] Parola, M., Bellomo, G., Robino, G., Barrera, G. & Dianzani, M. U. 4-Hydroxynonenal As a Biological Signal: Molecular Basis and Pathophysiological Implications. Antioxid Redox Signal 1, 255-284 (1999).

[0191] Qiao, H. et al. Protective effects of an anti-4-HNE monoclonal antibody against liver injury and lethality of endotoxemia in mice. Eur J Pharmacol 950, 175702 (2023).

[0192] Singh, P., Parajuli, N., Mayeux, P. R., & MacMillan- Crow, L. A.

[0193] (2016). Renal Mitochondrial Lipid Peroxidation during Sepsis. Journal of kidney, 2(1), 116.

[0194] Smathers, R. L. et al. Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics. PLoS One 7, e38459 (2012).

[0195] Sonowal, H. & Ramana, K. V. 4-Hydroxy-Trans-2-Nonenal in the Regulation of Anti-Oxidative and Pro-Inflammatory Signaling Pathways. Oxid Med Cell Longev 2019, 1-17 (2019).

[0196] Spitz, D. R., Sullivan, S. J., Malcolm, R. R. & Roberts, R. J.

[0197] Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal. Free Radic Biol Med 11, 415-423 (1991).van der Slikke, E. C. et al. Association between oxidized nucleobases and mitochondrial DNA damage with long-term mortality in patients with sepsis. Free Radic Biol Med 179, 156-163 (2022).

[0198] Wiesel, P., Patel, A. P., DiFonzo, N., Marria, P. B., Sim, C. U., Pellacani, A., Maemura, K., LeBlanc, B. W., Marino, K., Doerschuk, C. M., Yet, S. F., Lee, M. E., & Perrella, M. A. (2000). Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase- 1-deficient mice. Circulation, 102(24), 3015-3022.

[0199] Wu, H., & Pan, J. (2023). Correlation between serum 4-HNE and lactic acid levels and disease status in patients with severe pneumonia and its diagnostic value and prognostic evaluation. American journal of translational research, 15(3), 1913-1920

[0200] Zhang K, Zhang X, Ding W, et al. National Early Warning Score does not accurately predict mortality for patients with infection outside the intensive care unit: a systematic review and meta-analysis. Front Med 8, 704358 (2021)

Claims

47Claims1. A method for diagnosing sepsis in a subject, said method comprising: (a) detecting 4-HNE (4-hydroxy-2-nonenal) protein adducts in (i) CD14+ monocytes and / or (ii) CD3+ T cells in a sample obtained from the subject; (b) comparing the level of 4-HNE protein adducts in CD 14+ monocytes and / or CD3+T cells to that of a reference value obtained from non-sepsis control subjects; and(c) providing a diagnosis wherein a reduced level of 4-HNE adducts in CD 14+ monocytes and / or CD3+T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

2. The method according to claim 1, wherein at least a 10% decrease in the level of 4-HNE adducts in CD 14+ monocytes relative to the reference value and / or at least a 1.8-fold decrease in the level of 4-HNE adducts in CD3+T cells relative to the reference value indicates that the subject suffers from sepsis or is at risk of developing sepsis.

3. A method for diagnosing sepsis in a subject, said method comprising: (a) detecting 4-HNE (4-hydroxy-2-nonenal) protein adducts in (i) total peripheral blood leukocytes and in one or both of (ii) CD14+ monocytes and (iii) CD3+T cells in a sample obtained from the subject;(b) Calculating the ratio of 4-HNE protein adducts in CD 14+ monocytes to the 4-HNE protein adducts in peripheral blood leukocytes C’CD14 / total”) and / or the ratio of 4-HNE protein adducts in CD3+T cells to the 4-HNE protein adducts in peripheral blood leukocytes C’CD3 / total”) of the subject; and(c) using the one or more ratio(s) calculated under (b) to provide a diagnosis, wherein a ratio of CD14 / total below 2.7 and / or a ratio of CD3 / total48below 1.4 indicates that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

4. The method according to claim 3, further comprising comparing the level of 4-HNE protein adducts in peripheral blood leukocytes of the subject to that of a reference value, wherein an elevated level relative to the reference value is a further indicator that the subject suffers from sepsis or is at risk of developing (severe) sepsis.

5. The method according to claim 4, wherein said elevated level is at least a 6-fold, preferably at least a 8-fold, more preferably at least a 10-fold increase in 4-HNE protein adducts in peripheral blood leukocytes relative to the reference value.

6. The method according to any one of the preceding claims, comprising detecting 4-HNE protein adducts using a specific binding partner directed against 4-HNE, preferably an aptamer or antibody directed against 4-HNE.

7. The method according to any one of the preceding claims, wherein the sample is a blood sample comprising leukocytes, preferably wherein the sample is a buffy coat sample or a whole blood sample.

8. The method according to any one of the preceding claims, further comprising the use of a clinical scoring, preferably the National Early Warning Score (NEWS) scoring, more wherein the subject has a NEWS scoring < 8, preferably < 6, more preferably < 5.

9. The method according to any one of the preceding claims, further comprising determining the level of one or more cytokines, preferably selected from the group consisting of IL- 10, IL-6, and TNF-a.4910. The method according to any one of the preceding claims, wherein the method is performed using flow cytometry.

11. The method according to claim 10, comprising the steps of:- contacting an isolated sample from the subject with antibodies against one or both of cell surface CD3 and CD 14 followed by fixating and permeabilizing the cell;- staining the permeabilized cells with an antibody against 4-HNE;- subjecting the stained sample to flow cytometry and gating for CD 14+ monocytes and / or CD3+T cells;- determining the 4-HNE protein adduct level in CD14+ monocytes and / or CD3+ T cells and, optionally, in total peripheral blood leukocytes.

12. The method according to any one of claims 1 to 9, wherein the method is performed in a lateral flow assay format.

13. The method according to any one of the preceding claims further comprising selecting the subject for treatment with an anti-sepsis treatment, preferably wherein said anti-sepsis treatment comprises one or more of antibiotics, vasopressors and the use of intravenous fluids.

14. A diagnostic device comprising means for detecting 4-HNE protein adducts and means for capturing CD 14+ monocytes and / or CD3+T cells.

15. The device according to claim 14, wherein the diagnostic device is a point-of-care (PoC) test device.

16. The device according to claim 14 or 15, wherein the means for detecting 4-HNE protein adducts comprise aptamers, antibodies or antigenbinding fragments thereof capable of binding 4-HNE.5017. The device according to any one of claims 14 - 16, wherein the means for detecting 4-HNE protein adducts are conjugated to a detectable label, preferably selected from fluorochromes, gold nanoparticles (AuNPs), magnetic beads, quantum dots, enzymes, colloidal particles, or other detectable moieties.

18. The device according to any one of claims 14 - 17, wherein the means for capturing CD 14+ monocytes and / or CD3+T cells are antibodies or antigen-binding fragments thereof.

19. The device according to any one of claims 14 - 18, wherein the means for capturing CD 14+ monocytes and / or CD3+T cells are immobilized.

20. The device according to any one of claims 14 - 19, wherein the means for detecting 4-HNE protein adducts are in a dispersed or mobile phase.

21. The device according to any one of claims 14 - 20, comprising:an elongated housing defining a test strip holder containing a lateral flow test strip comprising means for detecting 4-HNE protein adducts, and antibodies against one or both of CD3 and CD 14,a fluid sampling chamber provided with an opening connecting the fluid sampling chamber with the test strip holder, anda viewing window in the elongate housing allowing reading of one or more portions of the lateral flow test strip.

22. The device according to any one of claims 14-21, comprising a conjugation zone comprising means for detecting 4-HNE protein adducts and a capturing zone comprising antibodies against one or both of CD3 and

Images