Method for determining whether a subject is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation

Abstract

A method for determining whether a subject is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease is disclosed. The method may comprise determining a quantitative value of lipid droplets in cells obtained from a biological sample of the subject, and / or a quantitative value of the ability of the cells to take up a lipoprotein; wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease.

Description

[0001] METHOD FOR DETERMINING WHETHER A SUBJECT IS AT RISK OF DEVELOPING OR HAVING INFLAMMATORY LEUKOCYTES AND / OR ATHEROSCLEROTIC PLAQUE INFLAMMATION

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method for determining whether a subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease .

[0004] BACKGROUND

[0005] Cardiovascular disease, including heart attack and stroke, is the leading cause of death worldwide . Several lipid lowering drugs exist in the market and more are being developed. These drugs can lower LDL-C, Apo-B containing lipoproteins, triglycerides and lipoprotein (a) . Furthermore, novel drugs for reducing specific atherogenic lipid species such as e . g. ceramides are in development .

[0006] Currently, the main focus for reducing cardiovascular risk is on lowering low density lipoprotein cholesterol . This may continue to be at the core of cardiovascular risk prevention, but additional lipoproteins and lipid species such as lipoprotein (a) (Ip (a) ) are receiving increased attention. This means that in the future treatment decisions will be more complex with combating LDL-C, triglycerides and lipoprotein (a) and potentially other lipid species .

[0007] A core question remains cardiovascular risk, as it might not be cost efficient to prescribe a complex repertoire of lipid lowering therapies to all patients . Therefore, better tools for cardiovascular risk assessment are required to define patient groups who are in need of more extensive lipid-lowering treatment approaches .

[0008] SUMMARY

[0009] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify keyfeatures or essential features of the claimed subj ect matter, nor is it intended to be used to limit the scope of the claimed subj ect matter .

[0010] A method for determining whether a subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease is disclosed. The method may comprise determining a quantitative value of lipid droplets in cells obtained from a biological sample of the subj ect, and / or a quantitative value of the ability of the cells to take up a lipoprotein; wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are included to provide a further understanding of the described embodiments and constitute a part of this specification, illustrate various advantageous features and examples of their combinations . In the drawings :

[0013] Figure 1 : Schematic overview of different sample collection and PBMC processing steps in combination with the semiautomated analysis flow to quantify lipid droplet abundance and lipoprotein uptake in PBMC cell samples .

[0014] Figure 2A: Schematic presentation of the quantification of lipid droplet abundance in white blood cells by determining the number of lipid droplets (LD-No) , lipid droplet size, (LD-size) the total cellular lipid droplet area (LD-Area) and the proportion of lipid droplet positive cells (LD-Pos) .

[0015] Figure 2B: Fluorescent picture of lipid droplets in white blood cells .

[0016] Figure 3 : Quantification of lipid droplet abundance in monocytes in lipid-poor conditions for subj ects with low (<20 nmol / 1) , intermediate (20-149 nmol / 1) , and high lipoprotein (a) (>=150 nmol / 1) , together with the average lipoprotein (a) concentration, LDL-C concentration and triglyceride concentration for the same subj ect groups .Figure 4 : Quantification of lipid droplet number, lipid droplet size and total lipid droplet area in lipid droplet-positive monocytes for individuals with low (<20 nmol / 1) , intermediate (20— 149 nmol / 1) and high (>=150 nmol / 1) circulating lipoprotein (a) concentration. As well as percent lipid-droplet positive monocytes for the same subj ect groups .

[0017] Figure 5A: Lipid droplet abundance in monocytes quantified in lipid poor conditions for subj ects with low (<20 nmol / 1) , intermediate (20-149 nmol / 1) and high (>=150 nmol / 1) lipoprotein (a) for individuals with LDL-C below or above 3.5 mmo 1 / 1 .

[0018] Figure 5B: Visualisation of LDL-C as mmol / 1 for the same patient groups as in A.

[0019] Figure 6 : Variability of monocyte lipid droplet numbers plotted against Lp (a) concentration, highlighting that individual with intermediate Lp (a) and low Lp (a) values can have high lipid droplet abundance in monocytes .

[0020] Figure 7 : Monocyte lipid droplet accumulation in two patients with familial chylomicronemia syndrome (FCS) . From left to right : Monocyte lipid droplet number, average monocyte lipid droplet size, and total lipid droplet area per monocyte .

[0021] Figure 8 : Lipid droplet accumulation in CD16-positive monocytes in two patients with familial chylomicronemia syndrome (FCS) . From left to right : Monocyte lipid droplet number, average monocyte lipid droplet size, and total lipid droplet area per monocyte .

[0022] DETAILED DESCRIPTION

[0023] A method for determining whether a subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease is disclosed.

[0024] The method may comprise determining a quantitative value of lipid droplets (LDs) in cells obtained from a biological sample of the subj ect, and / or a quantitative value of the ability of the cells to take up a lipoprotein;wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having the inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .

[0025] Lipid accumulation in circulating monocytes may be linked to increased arterial wall adhesion and transmigration as well as inflammation. Therefore, quantification of lipid droplet abundance and lipoprotein uptake in cells such as leukocytes offers new opportunities to provide additional insight into the progression and development of atherosclerosis .

[0026] Not to be bound by theory, lipid droplet accumulation and / or lipoprotein uptake may be more pronounced e . g. in e . g. CD16-positive monocytes which are known to be pro-inflammatory. For example, such monocytes may produce more pro-inflammatory cytokines such as TNFalpha, IL-lbeta and others . This means that they may stimulate an immune response and e . g. activate and attract other cells . Moreover, such monocytes might express surface markers which enable adhesion to the cells of the arterial wall enabling trans-migration into atherosclerotic plaques . Combined with the fact that they may accumulate in atherosclerotic plaques (lesions) , they may drive pro-inflammatory cell recruitment to atherosclerotic plaques and lead to inflammation within the plaques . As a result of processes relating to the inflammation, plaques might be more prone for rupture, may grow faster and may accelerate and / or increase the risk for developing atherosclerosis and / or cardiovascular disease . The growth of atherosclerotic plaques may in the end determine how likely it is to rupture and lead to cardiovascular disease, e . g. heart attack or stroke .

[0027] The inflammatory leukocytes may comprise or be e . g. inflammatory monocytes and / or lymphocytes . In this case, the inflammatory leukocytes may comprise or be, in particular, circulating foamy monocytes . Such circulating foamy monocytes may display lipid storage, e . g. lipid droplets or more lipid droplets within the cell (as compared e . g. to the majority of monocytes in the subj ect) . However, other leukocytes may also exhibit inflammatorychanges and be associated with the atherosclerotic plaque inflammation .

[0028] The increase in inflammatory leukocytes and / or in atherosclerotic plaque inflammation, atherosclerosis, and / or the cardiovascular disease may be mediated at least partially by an increase in foamy monocytes, in particular in circulating foamy monocytes, in the subj ect, and / or by an accumulation of foamy monocytes in atherosclerotic plaques in the subj ect . The circulating foamy monocytes may be understood as referring to monocytes which are present in blood. The circulating foamy monocytes may display lipid storage, e . g. quantified through the detection of cytoplasmic lipid droplets . A higher lipid storage in circulating monocytes may link to a higher pro-inflammatory burden of a subj ect . Any references to foamy monocytes in this specification may be understood, at least in some embodiments, as referring to circulating foamy monocytes .

[0029] Circulating foamy leukocytes may be understood as referring to leukocytes which can be detected in blood, such as monocytes, with an elevated lipid droplet content, i . e . an elevated or increased quantitative value of lipid droplets . In other words, the circulating foamy leukocytes may contain accumulated lipid droplets .

[0030] Circulating foamy leukocytes, such as circulating foamy monocytes, may leave the circulation and accumulate in atherosclerotic plaques, in particular in those in which inflammation occurs . The exact mechanisms and causal relationships may be highly complex and are not necessarily fully known yet . Further, in hypertriglyc-eridemic subj ects, lipoproteins may be easily taken up by monocytes and other leukocytes .

[0031] Foamy leukocytes, such as foamy monocytes, may express adhesion markers which make them more prone to adhere to the cells of the arterial wall and then trans-migrate into the atherosclerotic plaque and may thus contribute to plaque growth.

[0032] Therefore there may be a desire or need to recommend medication in order to reduce the amount of foamy leukocytes, such as foamy monocytes, in the subj ect, e . g. in the circulation of the subj ect . Such medication may be recommended even if the subject might not otherwise be a candidate for a lipid-lowering therapy,for example if they do not exhibit a high concentration of a lipoprotein, component of a lipoprotein, or a lipid that would otherwise be a criterium for recommending the lipid-lowering therapy.

[0033] The quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein may be indicative of the risk of an increased prevalence of foamy leukocytes (e . g. circulating foamy leukocytes, such as foamy monocytes) in the subj ect .

[0034] Current methods to quantify lipid droplet abundance are not direct lipid droplet readouts which measure lipid droplets indirectly due to morphological changes in cells ( flow cytometry side scatter readout) . More precise ways of quantifying lipid droplets may be work intensive, require skilled personnel and specialized equipment . Furthermore, these quantifications may have been done only with freshly isolated white blood cells . Consequently, the current methods do not enable application in a diagnostic setting in which a high throughput of samples and processing of a large set of samples in centralized facilities would be important .

[0035] With the present method, it may be possible to measure leukocyte lipid droplet abundance in a scalable fashion and more automated fashion. Furthermore, it is demonstrated that frozen cell samples can be processed and that cell samples can be processed after 24 to 48 h. The method may enable systematic quantification of leukocyte lipid droplet abundance in a setup which is relevant for diagnostic testing.

[0036] With the present method, it may be possible to identify subj ects who may be subj ect to a risk of developing cardiovascular disease and / or atherosclerosis, but who would not necessarily otherwise be considered as candidates for lipid lowering therapies . With the method, it can be determined whether the subj ect could benefit from a lipid lowering therapy, such as lipoprotein (a) lowering therapy, triglyceride-lowering therapy, and / or cholesterol-lowering therapy, even if they might not necessarily exhibit high cholesterol, lipoprotein (a) or triglyceride concentration ( s ) . Their risk of cardiovascular disease and / or atherosclerosis may thus be reduced.The method may be a method for determining whether the subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having the inflammatory leukocytes and / or atherosclerotic plaque inflammation .

[0037] The method may be a method for determining whether the subj ect is at risk of developing or having a disease or condition associated with and / or resulting from the inflammatory leukocytes and / or atherosclerotic plaque inflammation, wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having the disease or condition associated with and / or resulting from the inflammatory leukocytes and / or atherosclerotic plaque inflammation .

[0038] The method may be a method for determining whether the subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation, wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having the inflammatory leukocytes and / or atherosclerotic plaque inflammation .

[0039] The method may be a method for determining whether the subj ect is at risk of developing or having atherosclerosis and / or a cardiovascular disease, wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of atherosclerosis and / or a cardiovascular disease .

[0040] The method may be a method for determining whether the subj ect is at risk of developing atherosclerosis in an accelerated manner, wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the risk ofdeveloping atherosclerosis in an accelerated manner . In other words, the method may be a method for determining whether the subj ect is at risk of accelerated development of atherosclerosis, wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of accelerated development of atherosclerosis .

[0041] The method may be a method for mitigating the risk of the subj ect developing or having atherosclerosis and / or cardiovascular disease .

[0042] The cardiovascular disease may comprise or be at least one of coronary artery disease, such as angina or myocardial infarction, stroke, heart attack, cerebrovascular disease, or peripheral artery disease .

[0043] The subj ect may exhibit an elevated concentration of at least one of a component of a lipoprotein, a lipoprotein, or a lipid. The concentration may be elevated in the blood, for example in plasma or serum. In other words, the concentration of the at least one of a component of a lipoprotein, a lipoprotein, or a lipid may be elevated in the subj ect, for example in the blood (e . g. blood plasma or serum) of the subj ect .

[0044] The at least one of a component of a lipoprotein, a lipoprotein, or a lipid may comprise e . g. lipoprotein particles, lipoprotein (a) , LDL (low-density lipoprotein) , HDL (high-density lipoprotein) , VLDL (very low density lipoprotein) , IDL (intermediate density lipoprotein) , chylomicron particles, and / or any subgroup thereof, a triglyceride-rich lipoprotein, a Apo-B containing lipoprotein, cholesterol, a cholesterol ester, a triglyceride, a phospholipid, a ceramide, a sphingolipid, or another lipid species .

[0045] The subj ect may exhibit an elevated concentration of a component of a lipoprotein. The component of a lipoprotein may comprise or be e . g. lipoprotein (a) , cholesterol, a cholesterol ester, a triglyceride, a phospholipid, a ceramide, a sphingolipid, and / or another lipid species . The term "a triglyceride" may be understood as encompassing also triglycerides, or total triglycerides .The subj ect may exhibit an elevated concentration of a lipid. The lipid may comprise or be e . g. a triglyceride, cholesterol, a cholesterol ester, a phospholipid, a ceramide, a sphingolipid, and / or another lipid species .

[0046] The subj ect may exhibit an elevated concentration of a lipoprotein. The lipoprotein may comprise or be e . g. lipoprotein particles, lipoprotein (a) , LDL (low-density lipoprotein) , HDL (high-density lipoprotein) , VLDL (very low density lipoprotein) , IDL (intermediate density lipoprotein) , chylomicron particles, a triglyceride-rich lipoprotein, a Apo-B containing lipoprotein, and / or any subgroup thereof, for example lipoprotein (a) (which is an LDL subgroup or variant) .

[0047] The lipoprotein may comprise or be e . g. an ApoB-contain-ing lipoprotein.

[0048] The subj ect may exhibit an elevated concentration of a lipoprotein .

[0049] The subj ect may exhibit an elevated concentration of a lipid, for example triglycerides and / or phospholipids .

[0050] As a skilled person will understand, the subj ect may exhibit an elevated concentration of one or more of the a component of a lipoprotein, a lipoprotein, or a lipid.

[0051] The "elevated" concentration of the at least one of a component of a lipoprotein, a lipoprotein, or a lipid may depend on the exact component of a lipoprotein, lipoprotein, or lipid.

[0052] What is considered an elevated concentration, and guidelines concerning the same, may vary e . g. in different countries . What is considered an elevated concentration may also depend e . g. on data available and on the opinion of the international community of physicians and researchers working in the field. It may thus be subj ect to change, for example when new data becomes available . A skilled person may thus be able to deduce, for a given component of a lipoprotein, lipoprotein, or lipid, and at a given time, what an elevated concentration therefor may be .

[0053] The at least one of a component of a lipoprotein, a lipoprotein, or a lipid may comprise or be lipoprotein (a) and / or triglycerides . In other words, the subj ect may exhibit an elevated concentration of lipoprotein (a) and / or triglycerides .The at least one of a component of a lipoprotein, a lipoprotein, or a lipid may further comprise or be cholesterol, for example total cholesterol and / or LDL cholesterol (LDL-C) .

[0054] The at least one of a component of a lipoprotein, a lipoprotein, or a lipid may comprise or be lipoprotein (a) . In other words, the subj ect may exhibit an elevated concentration of lipoprotein (a) .

[0055] The "elevated" lipoprotein (a) concentration in the blood may be e . g. a concentration of at least about 7 mg / dl or 16 nmol / 1, or a concentration in the range of about 16 - 5000 nmol / 1.

[0056] In some embodiments, the subj ect may exhibit an intermediate or high concentration of lipoprotein (a) in the blood.

[0057] As indicated above, what is considered an "intermediate" or "high" concentration of lipoprotein (a) may vary and may be subj ect to change, and the skilled person is able to deduce what such a concentration may be .

[0058] The "intermediate" concentration of lipoprotein (a) may be e . g. a concentration in the range of about 16 - 75 nmol / 1. Additionally or alternatively, the "intermediate" concentration of lipoprotein (a) may be e . g. a concentration in the range of about 16 nmol / 1 - 50 nmol / 1, or about 16 nmol / 1 - 100 nmol / 1, or about 16 nmol / 1 - 125 nmol / 1, or about 16 nmol / 1 - 150 nmol / 1.

[0059] Also subj ects exhibiting an intermediate concentration of lipoprotein (a) in the blood may exhibit a relatively high abundance of circulating foamy monocytes .

[0060] The "high" concentration of lipoprotein (a) may be e . g. a concentration of at least about 75 nmol / 1, or a concentration in the range of about 75 nmol / 1 - 5000 nmol / 1. Additionally or alternatively, the "high" concentration of lipoprotein (a) may be e . g. a concentration of at least about 50 nmol / 1, or at least about 100 nmol / 1, or at least about 125 nmol / 1, or at least about 150 nmol / 1. Additionally or alternatively, the "high" concentration of lipoprotein (a) may be e . g. a concentration in the range of about 50 - 5000 nmol / 1, or about 100 - 5000 nmol / 1, or about 125 - 5000 nmol / 1, or about 150 - 5000 nmol / 1.

[0061] A method for determining whether the subj ect could benefit from a lipoprotein ( a) lowering medication is also disclosed.The method may thus be a method for determining whether the subj ect could benefit from a lipoprotein (a) lowering medication;

[0062] wherein the subj ect has an elevated lipoprotein (a) concentration in the blood;

[0063] wherein the method comprises determining the quantitative value of lipid droplets in the cells obtained from a biological sample of the subj ect, and / or the quantitative value of the ability of the cells to take up the lipoprotein; and

[0064] wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of whether the subj ect could benefit from the lipoprotein (a) lowering medication.

[0065] In this context, the elevated lipoprotein (a) concentration in the blood may be an intermediate or high concentration of lipoprotein (a) in the blood. In other words, the subj ect may exhibit an intermediate or high concentration of lipoprotein (a) in the blood.

[0066] In this context the subj ect may have an intermediate lipoprotein (a) concentration and would usually not receive lipoprotein (a) medication, as the subj ect is not contained in the high Ip (a) group . It may however be desirable to recommend Ip (a) lowering therapy to selected subj ects from the intermediate Ip (a) range, because they may exhibit a high amount of circulating foamy monocytes, even though they are below the high Ip (a) threshold.

[0067] The at least one of a component of a lipoprotein, a lipoprotein, or a lipid may comprise or be triglycerides . In other words, the subj ect may have an elevated concentration of triglycerides in the blood.

[0068] The "elevated" concentration of triglycerides in the blood may be e . g. a concentration of at least about 100 mg / dl, or a concentration in the range of about 100 mg / dl - 10000 mg / dl . Higher concentrations might also be contemplated.

[0069] Triglycerides may be temporarily elevated in the blood e . g. if the subj ect has recently eaten a meal high on fats . Such temporary elevation may however not be a reason to consider triglyceride lowering therapy. The subj ect may have a chronicallyelevated concentration of triglycerides in the blood. In other words, the elevated concentration of triglycerides in the blood is not temporary. The concentration of the triglycerides in the blood may, in some embodiments, be measured after fasting, for example after overnight fasting.

[0070] A method for determining whether the subj ect could benefit from a triglyceride lowering medication is also disclosed.

[0071] The method may thus be a method for determining whether the subj ect could benefit from a triglyceride lowering medication;

[0072] wherein the subj ect has an elevated concentration of triglycerides in the blood;

[0073] wherein the method comprises determining the quantitative value of lipid droplets in cells obtained from the biological sample of the subj ect, and / or the quantitative value of the ability of the cells to take up a lipoprotein; and

[0074] wherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of whether the subj ect could benefit from the triglyceride lowering medication.

[0075] In this context, the subj ect may have an intermediate concentration of triglycerides in the blood. The "intermediate" concentration of triglycerides in the blood may be e . g. a concentration in the range of 50 mg / dl - 500 mg / dl, or e . g. in the range of 150 - 500 mg / dl .

[0076] In this context the subj ect may have an intermediate concentration of triglycerides and would usually not receive triglyceride lowering therapy, because the concentration of the triglycerides would not be sufficiently high to warrant the therapy. It may however be desirable to recommend triglyceride lowering therapy to selected subj ects from the intermediate triglyceride concentration range based on the method according to the one or more embodiments described in this specification, because they may exhibit a high amount of circulating foamy monocytes, even though they are below the high triglyceride concentration threshold.

[0077] The method may further comprise providing a treatment recommendation for lipid lowering therapy, for example a lipid lowering medication. The method may further comprise providing atreatment recommendation for an optimal lipid lowering therapy, for example an optimal lipid lowering medication.

[0078] The lipid lowering therapy may comprise lipoprotein (a) lowering therapy, triglyceride lowering therapy, cholesterol lowering therapy, and / or other dyslipidemia therapy. In other words, the method may further comprise providing a treatment recommendation for lipoprotein (a) lowering therapy, for triglyceride lowering therapy, for cholesterol lowering therapy, and / or other dyslipidemia therapy. The treatment recommendation may comprise or be e . g. recommendation to administer a suitable therapy or medication .

[0079] For example, the method may further comprise providing a treatment recommendation for lipoprotein (a) lowering therapy, such as lipoprotein (a) lowering medication.

[0080] Additionally or alternatively, the method may further comprise providing a treatment recommendation for triglyceride lowering therapy, such as triglyceride lowering medication.

[0081] Additionally or alternatively, the method may further comprise providing a treatment recommendation

[0082] for cholesterol lowering therapy, such as cholesterol lowering medication .

[0083] The treatment recommendation may be provided on the basis of the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein, and on the basis of the at least one of a component of a lipoprotein, a lipoprotein, or a lipid, the concentration of which is / are elevated in the subj ect .

[0084] The cholesterol lowering therapy, such as medication, may comprise or be e . g. administration of a statin, ezetimibe, PCSK9 inhibitor, bempedoic acid, obicetrapib, a bile acid sequestrant, apheresis, an omega-3 fatty acid, such as eicosapentaenoic acid (EPA) , and / or icosapent ethyl ( IPE) , AP0C3 (Apolipoprotein C-III ) inhibitor therapy AP0C4 inhibitor therapy, ANGPTL3 inhibitor therapy, ANGPTL3 / 8 inhibitor therapy, ANGPTL4 inhibitor therapy, a GLP1 agonist, a GIP / GLP-1 dual agonist, a GIP / GLP-l / GCG triple agonist, a FGF21 analogous and / or multi-receptor agonist ( FGF21 / GLP-l / Glucagon) (e . g. through chemical molecules, RNA therapeutics, gene silencing therapies (e . g. siRNA, antisenseoligonucleotide (ASO) ) , gene editing, a cholesterol lowering antibody therapy, and / or cholesterol lowering therapy involving a peptide, protein, chemical molecule mimetic or analog capable of lowering cholesterol levels) .

[0085] The cholesterol lowering therapy may be therapy intended to specifically lower cholesterol concentration in the subj ect .

[0086] The triglyceride lowering therapy, such as triglyceride lowering medication, may comprise or be e . g. administration of niacin, an omega-3 fatty acid, EPA, IPE, APOC3 (Apolipoprotein C-III ) inhibitor therapy AP0C4 inhibitor therapy, ANGPTL3 inhibitor therapy, ANGPTL3 / 8 inhibitor therapy, ANGPTL4 inhibitor therapy, a GLP1 agonist, a GIP / GLP-1 dual agonist, GIP / GLP-l / GCG triple agonist, a FGF21 analogous and / or multi-receptor agonist ( FGF21 / GLP-l / Glucagon) (e . g. through chemical molecules, RNA therapeutics, gene silencing therapies (e . g. siRNA, antisense oligonucleotide (ASO) ) , gene editing, antibody approaches and or peptide, protein, chemical molecule mimetics or analogs) .

[0087] The triglyceride lowering therapy may be therapy intended to specifically lower the concentration of triglycerides in the sub ect .

[0088] The lipoprotein (a) lowering therapy, such as medication, may comprise or be e . g. administration of an antibody, gene silencing (e . g. siRNA, ASO) , gene editing or chemical (small) molecule to modulate (or lower) circulating lipoprotein (a) , a PCSK9 inhibitor, obicetrapib, muvalaplin, olpasiran, zerlasiran and / or pelacarsen. Moreover, therapeutic approaches to target oxidized lipid components (e . g. OxPL) (e . g. CAL103) and pro-inflammatory lipid components (e . g. ceramides) .

[0089] The lipoprotein (a) lowering therapy may be therapy intended to specifically lower lipoprotein (a) concentration in the subj ect . The other dyslipidemia therapy, such as dyslipidemia medication, may comprise or be e . g. administration of a ceramide lowering therapy or medication.

[0090] The treatment recommendation may comprise e . g. a recommendation for a combination of different treatment strategies, for example for lipoprotein (a) lowering therapy, triglyceride lowering therapy, and cholesterol lowering therapy. For example, if the subj ect has a high quantitative value of the lipid droplets in thecells, and only elevated lipoprotein (a) concentration, the treatment recommendation may comprise lipoprotein (a) lowering therapy, but not other therapies . If the subj ect exhibits an elevated lipoprotein (a) concentration and an elevated cholesterol concentration, the treatment recommendation may comprise both lipoprotein (a) lowering therapy and cholesterol lowering therapy.

[0091] While the subj ect could benefit from the lipid lowering therapy according to one or more embodiments described in this specification, a skilled person is well aware that not necessarily all subj ects respond, or respond well, to a given therapy or medication. The subj ect could nonetheless benefit from the lipid lowering therapy, i . e . the risk of developing or having atherosclerosis and / or cardiovascular disease may be mitigated, if it is determined that it is likely that the lipid lowering therapy could, assuming that the subj ect would respond thereto, and / or in the absence of a reason not to expect the subj ect to respond thereto, reduce the risk of the subj ect to develop or have inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease .

[0092] In the context of this specification, the terms "therapy" and "treatment" may be understood as commonly in the art . At least in some embodiments, they may be used interchangeably.

[0093] The method may further comprise administering a treatment to the subj ect . The treatment may be any treatment described in this specification.

[0094] In some embodiments, the subj ect does not exhibit hypercholesterolemia .

[0095] Additionally or alternatively, in some embodiments, the subj ect may have a blood LDL cholesterol (LDL-C) level of 1.4 mmol / I or less .

[0096] The method may comprise determining the quantitative value of lipid droplets (LDs) in cells obtained from the biological sample of the subj ect .

[0097] The quantitative value of lipid droplets may comprise one or more quantitative values, i . e . at least one quantitative value, of lipid droplets .The determining of the quantitative value of the lipid droplets may comprise determining at least one of, i . e . one or more of, the following:

[0098] - the amount of the lipid droplets in the cells, - the relative amount of the lipid droplets in the cells, - the area (e . g. combined total area) of the lipid droplets in the cells,

[0099] - the intensity (e . g. combined total intensity and / or an average intensity, or overall lipid droplet intensity) of the lipid droplets in the cells,

[0100] - the number of the lipid droplets in the cells, - the number or cells comprising lipid droplets (as not all of the cells obtained from the biological sample necessarily contain lipid droplets) , or

[0101] - the proportion of cells comprising lipid droplets (e . g.

[0102] from all of the cells obtained from the biological sample) .

[0103] The quantitative value of the lipid droplets may be determined in naive cells; in cells incubated in a lipid-rich medium; and / or in cells incubated in a lipid-poor medium. In other words, the quantitative value of the lipid droplets may be determined in naive conditions; in lipid-rich conditions; and / or in lipid-poor conditions .

[0104] Naive cells may be understood as referring to cells that have not been subjected to, for example incubated in, any particular conditions or medium prior to determining the quantitative value of the lipid droplets .

[0105] The lipid-poor (or lipid starvation) conditions, or medium, may refer e . g. to a medium containing 5% lipoprotein deficient serum. Usually 10% fetal bovine serum is added to cell culture cells; the fetal bovine serum contains lipoproteins . Thus the lipid-poor medium may contain less lipoproteins than a medium containing 10 % fetal bovine serum. For example, the lipid-poor medium may comprise 50 % (w / w) or less, or 20 % (w / w) or less, of the lipoproteins contained in a medium containing 10 % fetal bovine serum. Lipoproteins may have been removed from the lipoprotein deficient serum. The lipid-poor conditions are a very good way to induce lipid starvation. As another example, the lipid-poor mediummay comprise only up to 1 % (w / w) fetal bovine serum instead of 10 % . The lipid-rich medium may thus be considered a complete medium, while the lipid-poor medium may be considered to be a lipoprotein depleted medium.

[0106] Quantitative values of the lipid droplets may be used to obtain a quantitative value of the lipid mobilization capability of the cells .

[0107] The quantitative value of the lipid mobilization capability of the cells may be determined e . g. by determining the quantitative value of lipid droplets (LDs) in the cells in complete medium relative to the quantitative value of lipid droplets in the cells in lipoprotein depleted medium. This may be done e . g. by calculating a ratio of the quantitative value of lipid droplets in the cells in complete medium relative to the quantitative value of lipid droplets in the cells in lipoprotein depleted medium. The term "complete medium" may be understood as referring to a cell culture medium containing all components required for the maintenance and / or growth of the cells . An example of a suitable complete medium is a medium containing 10% FBS ( fetal bovine serum) . An example of a lipoprotein depleted medium is a comparable medium (i . e . a medium comparable to a corresponding complete medium) that does not contain FBS but contains 5% LPDS (lipoprotein deficient serum) .

[0108] The quantitative value of lipid droplets (LDs) in the cells a may be determined by measuring the abundance of lipid droplets in intracellular lipid storage organelles . The cells may be immobilized e . g. to a plastic or glass substrates . Then the cells may be fixed and stained with a nuclear stain and a hydro-phobic fluorophore which accumulates inside lipid droplets . Then the cells may be subj ected to automatic imaging; software tools may be used to identify the cells and individual lipid droplets in each cell . This may allow for determining the exact number of cellular lipid droplets, their size and / or their fluorescent intensity. Details of the method have been described e . g. by Pfist-erer et al . , Nature Communications 2017 ; 8 : 14858.

[0109] However, other methods for determining the quantitative value of the lipid droplets may be contemplated, for example, biochemical determination of lipids under different conditions, orflow cytometry may be used to quantify LD intensities under the different conditions . Alternatively, the lipid mobilization may be measured by first exposing the cells to a high lipid load to stimulate a large number of lipid droplets and then switch to complete medium which contains less lipids, thereby monitoring lipid mobilization. With automated microscopy and image analysis it may be possible to achieve a more accurate quantification of lipid droplets . Flow cytometry could also be used to measure the cellular lipid droplet intensity, but not necessarily the number and the sizes of the lipid droplets . Nonetheless, the flow cytometry could be used to quantify intensity changes under different conditions and thus permit the calculation of lipid mobilization. Also, lipid droplets could be quantified by antibody stainings for a lipid droplet associated protein.

[0110] In the context of this specification, the term "lipid mobilization capacity" may be understood as referring to the capacity of the cells to utilize and mobilize stored lipids . In an embodiment, the term "lipid mobilization capacity" may be understood as referring to a ratio and / or fold difference of the quantitative value of lipid droplets, such as lipid droplet numbers, cellular lipid droplet area, and / or cellular lipid droplet intensity, in complete medium relative to the quantitative value of lipid droplets, such as lipid droplet numbers, cellular lipid droplet area, and / or cellular lipid droplet intensity, in lipoprotein depleted medium (e . g. in the same or comparable cells) . However, the quantitative value of the lipid mobilization capacity may, alternatively or additionally, be determined by determining the difference between lipid droplet numbers, cellular lipid droplet area, and / or cellular lipid droplet intensity in complete medium and in lipoprotein depleted medium.

[0111] The method may comprise comparing the quantitative value of the lipid droplets to a quantitative value of control cells or to a control value, wherein an increase in the quantitative value of the lipid droplets is indicative of an increased risk of developing or having the inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .The method may comprise determining the quantitative value of the ability of the cells to take up a lipoprotein.

[0112] In this context, the lipoprotein (i . e . the lipoprotein taken up by the cells) may comprise or be low-density lipoprotein (LDL) ; oxidized LDL; VLDL; oxidized VLDL; lipoprotein (a) ; and / or chylomicron particles . For example, the lipoprotein may comprise or be low-density lipoprotein (LDL) .

[0113] The lipid droplet accumulation may be linked to the ability of the cells to take up lipoproteins . For example, changes in lipoprotein uptake might explain why some people have higher lipid droplet abundance in leukocytes as compared to others .

[0114] The quantitative value of the ability of the cells to take up the lipoprotein, such as LDL, may be determined e . g. by contacting the cells with labelled lipoprotein particles, such as LDL particles; incubating the resulting mixture thereby obtainable for a period sufficient to allow cellular internationalization of the labelled lipoprotein particles; and determining the amount and / or the number of labelled lipoprotein particles internalized by the cells . Alternatively or additionally, the intensity of the labelled lipoprotein particles internalized by the cells and / or the quantification of organelles containing the labelled lipoprotein particles may be used to determine the quantitative value of the ability of the cells to take up the lipoprotein. The labelled lipoprotein particles may bind to a suitable receptor, for example an LDL receptor (or LDL receptors) , on the surface of the cells, such that a complex is formed by the labelled lipoprotein particles and the receptor . The complex may then be internalized into the cells . However, other methods capable of measuring the ability of the cells to take up the lipoprotein may be contemplated, for example methods utilizing ApoB (apolipoprotein B) or artificial lipoprotein molecules .

[0115] The method may comprise comparing the quantitative value of the ability of the cells to take up the lipoprotein to a quantitative value of control cells or to a control value, wherein an increase in the quantitative value of the ability of the cells to take up the lipoprotein is indicative of an increased risk of developing or having the inflammatory leukocytes and / oratherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .

[0116] In some embodiments, the method may comprise determining the quantitative value of lipid droplets (LDs) in the cells obtained from the biological sample of the subj ect, and the quantitative value of the ability of the cells to take up the lipoprotein .

[0117] The biological sample of the subj ect may be a blood sample or a sample derived from a blood sample, e . g. a buffy coat sample . Other biological samples may be contemplated, for example cell samples derived from tissues such as fibroblasts, keratinocytes and / or mesenchymal stem cells .

[0118] The cells (i . e . the cells obtained from the biological sample of the subj ect) may comprise or be leukocytes, such as peripheral blood mononuclear cells (PBMCs) , lymphocytes, monocytes, granulocytes, hematopoietic progenitor cells and / or neutrophils . In general, leukocytes may be considered to include lymphocytes, monocytes, granulocytes, neutrophils and hematopoietic stem cells . PBMCs may be obtained from the biological sample, such as a blood sample or buffy coat sample, e . g. by density gradient centrifugation. PBMCs may also be selected by positive or negative antibody selection techniques . The cells may, alternatively or additionally, comprise or be e . g. lymphocytes immortalized with Epstein-Barr virus (EBV lymphoblasts) ; neutrophils; and / or granulocytes .

[0119] The cells may comprise or be monocytes . Monocytes are well suited for the present method.

[0120] The cells may be isolated after immediately after blood collection or first shipped and / or transferred before isolation. In this case cell isolation may occur at least 3 h after the collection .

[0121] The cells may be cryopreserved.

[0122] The biological sample and / or the cells may be frozen and / or processed at least 3 hours after the biological sample has been collected. The biological sample and / or the cells may be processed e . g. by thawing and optionally isolating the cells . The quantitative value (s) may then be determined from the processed cells . The processing of the samples may involve transport ortransfer of the blood samples, followed by cell isolation, cryopreservation of the cells and then determining the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein.

[0123] The subj ect may be human, but the subj ect may, additionally or alternatively, be an animal, such as a mammalian, for example, a primate, a non-human primate, a dog, a cat, a horse, a sheep, a goat, a bovine, a rabbit, a pig and / or a rodent, such as a mouse or a rat, or any other species .

[0124] The subj ect may be generally healthy. In some embodiments, the subj ect has not been diagnosed with atherosclerosis and / or cardiovascular disease . In some embodiments, the subj ect does not receive a lipid lowering therapy, such as medication. In some embodiments, the subj ect does not receive any lipid lowering therapy, such as medication.

[0125] In some embodiments, the subj ect does not suffer from or carry an allele for familiar hypercholesterolemia .

[0126] The quantitative value of lipid droplets and / or the quantitative value of the ability of the cells to take up a lipoprotein may be determined using a semi-automated analysis pipeline .

[0127] Such a semi-automated analysis pipeline may include automated image and data analysis . The semi-automated analysis pipeline may comprise a high content imaging unit configured to perform an automated image and data analysis . The high content imaging unit may comprise an automated microscopy unit, such as an automated fluorescence microscopy unit . The automated microscopy unit may include e . g. automated wide-field fluorescence microscopy and / or confocal microscopy. High content imaging may allow better automation and / or flexibility. It may also reduce the time required for the method.

[0128] The semi-automated analysis pipeline may comprise a robotic platform.

[0129] However, other methods may, alternatively or additionally, be contemplated. For example, the method may be performed using flow cytometry and / or non-automated microscopy.

[0130] The cells may be immobilized on a surface . For example, they may be immobilized on the surface for high content imaging. The cells may be immobilized on the surface as a monolayer .Suitable surfaces may include e . g. plastic and / or glass surfaces, such as high-content imaging plates . The immobilization may allow for storage and repeated analyses (e . g. image acquisition) of the (same) cells after storage . It may allow for grouping and / or discriminating between two or more cell populations . The immobilization may also prevent cell aggregation and allow for automation of staining and / or imaging of the cells . Immobilized cells may be suitable for imaging at a subcellular resolution that may enable determining the quantitative amount of lipid droplets and / or internalized LDL particles . The immobilization may also allow for the determination of the subcellular localization of the lipid droplets and / or internalized lipoprotein particles .

[0131] The immobilized cells may adhere to the surface . To facilitate adhesion, the surface may be selected, treated and / or prepared such that it allows for improved adhesion. Suitable surfaces may be coated. Suitable coatings may include e . g. coatings that may facilitate cell adhesion to the surface, such as a Poly-D lysine (PDL) , Poly-L-lysine, collagen, and / or laminin coatings .

[0132] The cells may be grouped to two or more (i . e . at least two) cell populations, and / or one or more cell populations may be selected from the cells, and the quantitative value of the lipid droplets and / or the quantitative value of the ability to take up the lipoprotein may be determined for at least one of the cell populations separately from the other cell populations . For this, the cells may be immobilized on a surface . Immobilizing the cells may however not be necessary. For example, the cells may be grouped to two or more cell populations e . g. by an antibody staining using an antibody specific for a cell population or other type of staining capable of differentiating between the two or more cell populations, and / or by their morphological properties . For example, the at least one cell population may be a cell population exhibiting the highest signal to noise ratio (as compared to the other cell population ( s ) within the cells) . As another example, PBMC cells may be grouped to monocytes and lymphocytes (and optionally to other cell types) , and the quantitative value (s) may be determined separately for the monocytes, lymphocytes, and any other optional cell populations . Monocytes and lymphocytes may be discriminated from each other based on size, as monocytes may spreadout after adhesion to a surface, such as a coated imaging plate, resulting in an enlarged cytoplasmic region. However, at least in some embodiments, the quantitative value (s) determined separately for the monocytes, lymphocytes, and / or any other optional cell types or populations, may be combined and used e . g. in correlations .

[0133] However, grouping the cells to two or more cell populations may not be necessary for determining the quantitative value (s) . For example, the quantitative value (s) may be determined from an entire PBMC population (or a part thereof ) .

[0134] The quantitative value of the ability of the cells of the subj ect to take up the lipoprotein may be determined using labelled lipoprotein particles . However, other means which may allow for the determination of the ability of the cells of the subj ect to take up the lipoprotein may also be contemplated. The lipoprotein particles may be e . g. fluorescently or radioactively labelled. The lipoprotein particles may be preserved e . g. by freezing.

[0135] The quantitative value of the ability of the cells of the subj ect to take up the lipoprotein may be determined using lipoprotein particles preserved by snap freezing. The snap freezing may be done e . g. by lowering the temperature of the lipoprotein particles to a temperature of -70 °C or lower very quickly, for example for a time period of at least 1 s, or at least 5 s, or at least 10 s, or at least 30 s, or at least 1 min. After the snap freezing, the lipoprotein particles thus preserved may be stored e . g. at a temperature of about -70 to about -80 °C; however, they may also be stored at other temperatures, such as temperatures below about -10 °C or below about -20 °C, or in the range of about -10 °C to about -20 °C . The snap freezing and optionally storing the lipoprotein particles at a suitable temperature may prevent the aggregation or modification of the lipoprotein particles, which could alter the measurement of the quantitative value of the ability of the cells to take up the lipoprotein. Thus the lipoprotein particles may be stored longer and / or provide more reliable results .

[0136] The quantitative value (s) may be normalized using a standard comprising a mixture of cells obtainable from biological samples of a plurality of subj ects . The plurality of subj ects maycomprise or be e . g . at least 2 , or at least 3, or at least 4 , or at least 5, or at least 10 subj ects, or at least 100 subj ects . Thus it may be possible to obtain a standard that is more reproducible and / or more uniform than e . g. standards obtainable from a biological sample of a single person.

[0137] Large-scale control standards may be used to normalize the data . New control standards may be compared to previous control standards to allow comparison of samples over a long timeframe . The different overlapping control standards may be measured e . g. in 3 or more experiments to have enough values so as to compare them reliably. For example, this may be done by providing a mixed preparation of PBMCs from e . g. 4 or more individuals . These standards may be aliquoted and frozen so that each aliquot is sufficient for one experiment . For each test or experiment a new aliquot may be used. This may ensure that the standard is of the same or similar quality for each experiment . Once a new standard is made, the standard may be compared to a previous control standard, by quantifying e . g. lipoprotein uptake and lipid droplet abundance after treatment with control medium and lipoprotein depleted medium. The background of the cells may also be quantified. For example, only standards which are similar with regard to background, lipoprotein uptake and lipid droplet abundance to the previous standards may be considered as suitable standards for upcoming experiments . For long term comparability of experimental results, it may be beneficial to compare results obtained from one standard to results obtained with a different standard. Therefore, the different standards may be measured in multiple experiments to reduce effects from experimental variation.

[0138] Results obtainable by the method may be combined with other measures to improve the determination of the risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .

[0139] The method may further comprise calculating a risk score based on the quantitative value (s) .

[0140] Various other characteristics, measures or values may be included in the calculation of the risk score . Such other characteristics, measures or values may include e . g. one or more of thefollowing: age, gender, dietary habits, physical measurements such as body mass index (BMI ) , hip circumference, waist circumference, blood metabolites, circulating lipoprotein concentrations, genetic risk factors, family history and / or previous medical history. Such other characteristics, measures or values may be determined prior to, simultaneously, or after the biological sample is obtained from the subj ect . As a skilled person will understand, some of the characteristics, measures or values may be information collected e . g. using a questionnaire or clinical data collected earlier . Some of the characteristics, measures or values may be determined (or may have been determined) by biochemical or clinical diagnostic measurements and / or medical diagnosis .

[0141] EXAMPLES

[0142] Reference will now be made in detail to various embodiments, an example of which is illustrated in the accompanying drawings .

[0143] The description below discloses some embodiments in such a detail that a person skilled in the art is able to utilize the embodiments based on the disclosure . Not all steps or features of the embodiments are discussed in detail, as many of the steps or features will be obvious for the person skilled in the art based on this specification.

[0144] EXAMPLE 1

[0145] PBMC isolation: Blood or buffy coat samples were mixed 1 : 1 with phosphate buffered saline (PBS) including 2.5 mM EDTA (PBS-E) . The blood mixture was gently layered over Histopacque Premium ( 1.0073, for mononuclear cells) and centrifuged 40 min at 400 g. The PBMC cell layer was removed, transferred to a new 15 ml reaction tube and mixed with PBS-E . Cells were centrifuged at 400 g for 10 min and incubated in 2 ml of red blood cell lysis buffer for 1 min ( 155 mM NH4C1, 12 mM NaHCO3, 0 . 1 mM EDTA) . 10 ml of PBS-E was added and cells were pelleted and washed with PBS-E . Then cells were resuspended in 5 ml PBMC medium (RPMI-1640, penicil-lin / streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1 mM HEPES) , counted, pelleted and resuspended in freezing medium (70%PBMC-medium, 20% FBS, 10 % DMSO) and cryopreserved in liquid nitrogen .

[0146] Cell treatments, transfer to imaging plates and. fixation: Cryopreserved EBV lymphoblasts or PBMCs were thawed in PBMC medium, and centrifuged at 400 g for 10 min. The cells were resuspended in PBMC medium and transferred to wells of a 96 well plate containing FBS ( 10% final concentration) or LPDS (5% final concentration) and incubated for 24 h. Subsequently, cells were transferred to conical 96 well plates and centrifuged at 400 g for 10 min. Using a robotic platform (Opentrons, New York, USA) medium was removed and cells were resuspended in PBMC medium. Cells were centrifuged, automatically resuspended in PBMC medium and transferred to PDL coated 384 well high-content imaging plates (Corning) . After 30 min of incubation at 37 °C cells were automatically fixed with 4% paraformaldehyde in 250 mM HEPES, 1 mM CaC12, 100 pM MgC12, pH 7.4 and washed with PBS . For lipid droplet stainings, cells were directly transferred to PDL coated 384 well high-content plates, adhered, automatically fixed and washed with PBS .

[0147] Lipid droplet analyses : Cells were processed as described before (Pfisterer et al . , Nat Commun. 2017 ; 8 : 14858 ) with the following changes : Fixed cell samples were automatically stained with 1 pg / ml LD540 (Princeton BioMolecular Research) and 5 pg / ml DAPI .

[0148] 3D stacks of optical slices were acquired automatically with a PerkinElmer Opera Phenix High Content Imaging system with a 63x water immersion obj ective, NA 1 .15. Image stacks were automatically deconvolved either with a custom made Python tool based on the open-source tools PSF generator and deconvolution lab . Maximum intensity proj ections were made from the deconvolved image stacks with custom Python tools . These tools can be accessed via : https : / / github . com / lopaavol / Oputils . Automated quantification of lipid droplets was performed with CellProfiler and as described previously. (Pfisterer et al . , Nat Commun. 2017 ; 8 : 14858 , which is incorporated herein in its entirety) . Lipid droplet counts were normalized to the average of two control standards which were included in each imaging plate .

[0149] Data analysis: Python (Python Software Foundation, www.python. org) was used with the Pandas package (pandas . pydata . org) to analyse single cell data obtained fromCellProf iler . With a cytoplasm area below 115 pm2cells were classified as lymphocytes whereas cells with an area above 115 pm2were classified as monocytes . Statistical significance was assessed with SciPy (scipy. org) using the "ttest_inde" function which calculates a two-tailed p value for independent samples . Data visualization was performed with Matplotlib (matplotlib . org) and Seaborn (seaborn.pydata . org) .

[0150] EXAMPLE 2

[0151] Systematic quantification of leukocyte lipid droplet abundance in a form of a clinical application.

[0152] Lipid droplets may be quantified in various ways in research settings . In one approach lipid content is indirectly estimated by performing flow cytometry and using side scatter technology to detect lipid droplet accumulation in cells . However, side scatter readouts are not specific to lipid droplets and can be induced by other morphological changes . Furthermore, it does not enable quantification of individual lipid droplets in cells as well as their size and area . This approach therefore does not enable reliable quantification of leukocyte lipid droplet accumulation. In another approach monocytes are stained for lipid droplet stains and subj ected to confocal microscopy and quantification of lipid droplet positive cells . This approach is highly manual and requires skilled personnel . Moreover, only a small amount of cells is quantified and is highly subj ective to the researcher who is acquiring representative images with the microscopy and quantifying them. Consequently, both approaches are not suitable as a clinical application in which lipid droplet accumulation is utilized for diagnostic purposes . A semi-automated analysis platform is described herein, which platform can provide systematic quantification of different lipid droplet parameters from lymphocyte and monocyte cell populations, utilizing robotic tools, automated microscopy, automated image and automated data analysis (Figure 1 ) . The leukocyte quantifications are performed in a centralized laboratory and samples can be channelled into the analysis flow in 4 main ways . 1 ) Blood samples are collected at clinical sites, delivered to the service facility within 48 h for the isolation of peripheral blood mononuclear cells (PBMCs) and PBMCs arecryopreserved before processing. 2 ) Blood samples are collected at clinical sites, delivered to the service facility within 24 h for PBMC isolation and PBMCs are processed fresh. 3) Blood samples are collected at the service facility, PBMC isolation is performed immediately and cells are cryopreserved before analysis . 4 ) Blood samples are collected at the service facility, PBMC isolation is performed and PBMCs are immediately processed (Figure 2 ) .

[0153] For lipid droplet quantification PBMCs are either directly transferred to 384 well imaging plates or first incubated for 24 h in lipid-poor or rich conditions in 96 well plates . Robotic tools are used to transfer cells to 384 well high content imaging plates . Cells are adhered to the bottom of the plate by incubating the plates at 37 °C and 5% C02 for 45 min. Then cells are fixed and prepared for automated microscopy with a robotic platform. Lipid droplets can be visualized by different stains (e . g. LD540, BODIPY) , nuclei and cytoplasm are stained as well . Then 12 -18 image stacks each containing information of approximately 50 to 100 are acquired for nuclei, cytoplasm and lipid droplet stains per well . The lipid droplet channels are deconvolved and cells and lipid droplets are automatically quantified with an image analysis software . The quantification includes the number, size and total area of lipid droplets per cell as well as the number or percentage of lipid droplet positive cells (Figure 2 ) . The quantifications are performed for lymphocyte or monocyte-enriched populations but can be further refined by including surface expression markers such as CD16.

[0154] The described approach is amenable as a clinical application. Samples can be collected e . g. across Europe and transported by overnight delivery to a service facility for processing. More remote collection sites can be enabled by on site PBMC isolation, cryopreservation and sample delivery on dry ice . Moreover, the semi-automated analysis pipeline is less biased and more obj ective, providing more reproducible results . The automated aspects of the analysis platform ensure that samples can be processed in a couple of days enabling turnaround times which are similar to a genetic test . These aspects are important for clinical applications .EXAMPLE 3

[0155] Increased, monocyte lipid droplet abundance in individuals with intermediate and high Lp (a) .

[0156] Lipoprotein (a) (Lp (a) ) is an independent risk factor for cardiovascular disease . So far it is unclear whether individuals with elevated lipoprotein (a) display altered lipid metabolism in PBMCs . To address this questions we performed a systematic characterization of cellular lipid droplet abundance in lymphocyte and monocyte-enriched cell populations from 60 subj ects . These included 24 subj ects with low Lp (a) ( 0-19 nmol / 1) , 17 subj ects with intermediate Lp (a) (20-149 nmol / 1) and 19 subj ects with high Lp (a) (>=150 nmol / 1) . The normalized lipid droplet content in monocytes measured after incubation in lipid-poor conditions increased in a stepwise manner from the low Lp (a) group, to the intermediate and high Lp (a) groups (Figure 3) . Whilst the Lp (a) concentration in the high Lp (a) group was about five times higher than in the intermediate group, LDL-C and triglycerides were roughly similar . Indicating higher levels of Lp (a) have an important impact on monocyte lipid droplet abundance (Figure 3) .

[0157] EXAMPLE 4

[0158] Changes in lipid droplet parameters for subjects with intermediate and high Lp(a)

[0159] Besides the normalized lipid droplet (LD) number across all monocytes we also quantified the non-normalized number of lipid droplet number, size and area in lipid droplet-positive monocytes . Importantly, the number of lipid droplet-positive monocytes increases in a step-wise manner for subj ects with intermediate and very high Lp (a) . Moreover, LD number and LD area increased within lipid droplet-positive monocytes . This indicates that not only the number of lipid droplet-positive cells is increasing in subj ects but that cells also accumulate more lipid droplets . For lipid droplet-positive cells the increase in total lipid droplet area appears to be mostly drive by the increase in LD numbers as the size of LDs remained similar for subj ects with low, intermediate and high Lp (a) concentration.

[0160] EXAMPLE 5Contribution of elevated LDL- cholesterol and Lp (a) to monocyte lipid droplet accumulation.

[0161] To investigate whether elevated LDL-C can contribute to increased lipid droplet formation in subj ects with low, intermediate and high Lp (a) we divided the subj ects into low (<3 . 5 mmol / 1) and high LDL-C (>3.5 mmol / 1) . In each Lp (a) group monocyte lipid droplet numbers were higher the high LDL-C (Figure 5A) . Whilst the same pattern was observed for each Lp (a) group the difference was most pronounced for the low Lp (a) group (Figure 5 A) . Probably because of lower contributions from Lp (a) on monocyte LD numbers . LDL-C concentrations were twofold higher in the high LDL-C group as compared to the low LDL-C group but were similar for the different Lp (a) subgroups . The results highlight that both elevated LDL-C and elevated Lp (a) can contribute to increased lipid droplet accumulation in monocytes .

[0162] EXAMPLE 6

[0163] Inter individual variability of monocyte lipid droplet accumulation

[0164] We observed large variability in monocyte lipid droplet for individuals with similar Lp (a) concentration (Figure 6) . Some subj ects with intermediate Lp (a) displayed very high monocyte lipid droplet accumulation whilst some subj ects with high Lp (a) only contained moderate LD accumulation in monocytes (Figure 6) . This highlights that even though lipoprotein (a) is associated with increased monocyte LD accumulation, the effects are very different from person to person. Consequently, it is important to quantify lipid droplets in monocytes and not extrapolate them from Lp (a) or LDL-C .

[0165] EXAMPLE 7

[0166] Monocyte lipid droplet abundance in patients with familial chylomicronemia syndrome (FCS) patients

[0167] Familial chylomicronemia syndrome (FCS) is a genetic disorder which leads to high triglyceride levels in blood. Here we show for the first time that subj ects with FCS display lipid droplet accumulation in monocytes (Figure 7 ) . The total lipid dropletarea in monocytes increased up to three-fold which was driven by an increase in lipid droplet numbers per monocyte and an increase in the average lipid droplet size . Lipid droplet numbers increased in FCS patients as well as the lipid droplet size .

[0168] EXAMPLE 8

[0169] Profound, lipid droplet accumulation in CD- 16 positive monocytes in FCS patients.

[0170] GDI 6 monocytes are known to have pro-inflammatory activities with higher levels of pro-inflammatory cytokine production and enhanced tissue migration capability. We observed higher lipid droplet abundance in GDI 6 positive monocytes (Figure 8 ) for FCS patients as compared to the overall monocyte population (Figure 7 ) . Especially, lipid droplet numbers and size increased and consequently also the total lipid droplet area per monocyte . Interestingly we observed also high lipid droplet accumulation in CD16-positive monocyte from one control individual (Figure 8 ) . Again, indicating that probably large interindividual differences in lipid droplet accumulation exist for monocytes and CD16-positive monocytes .

[0171] While there have been shown and described and pointed out fundamental novel features as applied to preferred embodiments thereof, it will be understood that various omissions and substitutions and changes in the form and details of the devices and methods described may be made by those skilled in the art without departing from the spirit of the disclosure . For example, it is expressly intended that all combinations of those elements and / or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure . Moreover, it should be recognized that structures and / or elements and / or method steps shown and / or described in connection with any disclosed form or embodiments may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice .

[0172] The applicant hereby discloses in isolation each individual feature described herein and any combination of two ormore such features, to the extent that such features or combinations are capable of being carried out based on the present specification as a whole, in the light of the common general knowledge of a person skilled in the art, irrespective of whether such features or combinations of features solve any problems disclosed herein, and without limitation to the scope of the claims . The applicant indicates that the disclosed aspects / embodiments may consist of any such individual feature or combination of features . In view of the foregoing description it will be evident to a person skilled in the art that various modifications may be made within the scope of the disclosure .

[0173] It is obvious to a person skilled in the art that with the advancement of technology, the basic idea may be implemented in various ways . The embodiments are thus not limited to the examples described above; instead they may vary within the scope of the claims .

[0174] The embodiments described hereinbefore may be used in any combination with each other . Several of the embodiments may be combined together to form a further embodiment . A method, a product, an arrangement, or a use, disclosed herein, may comprise at least one of the embodiments described hereinbefore . It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments . The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages . It will further be understood that reference to ' an' item refers to one or more of those items . The term "comprising" or "including" is used in this specification to mean including the feature (s) or act (s) followed thereafter, without excluding the presence of one or more additional features or acts .

Claims

CLAIMS1. A method for determining whether a subj ect is at risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or a cardiovascular disease,wherein the subj ect exhibits an elevated concentration of at least one of a component of a lipoprotein, a lipoprotein, or a lipid;wherein the method comprises determining a quantitative value of lipid droplets in cells obtained from a biological sample of the subj ect, and / or a quantitative value of the ability of the cells to take up a lipoprotein; andwherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of the increased risk of developing or having inflammatory leukocytes and / or atherosclerotic plaque inflammation; atherosclerosis; and / or the cardiovascular disease .

2. The method according to claim 1 , wherein the at least one of a component of a lipoprotein, a lipoprotein, or a lipid comprises or is lipoprotein (a) and / or triglycerides .

3. The method according to claim 1 or 2, wherein the subj ect exhibits an intermediate or high concentration of lipoprotein (a) in the blood.

4. The method according to any one of claims 1 - 3, wherein the method is a method for determining whether the subj ect could benefit from a lipoprotein (a) lowering medication;wherein the subj ect has an elevated lipoprotein (a) concentration in the blood;wherein the method comprises determining the quantitative value of lipid droplets in the cells obtained from a biological sample of the subj ect, and / or the quantitative value of the ability of the cells to take up the lipoprotein; andwherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cellsto take up the lipoprotein is / are indicative of whether the subj ect could benefit from the lipoprotein (a) lowering medication.

5. The method according to any one of claims 1 - 4, wherein the method is a method for determining whether the subj ect could benefit from a triglyceride lowering medication;wherein the subj ect has an elevated concentration of triglycerides in the blood;wherein the method comprises determining the quantitative value of lipid droplets in cells obtained from the biological sample of the subj ect, and / or the quantitative value of the ability of the cells to take up a lipoprotein; andwherein the quantitative value of the lipid droplets in the cells and / or the quantitative value of the ability of the cells to take up the lipoprotein is / are indicative of whether the subj ect could benefit from the triglyceride lowering medication.

6. The method according to any one of claims 1 - 5, wherein the method further comprises providing a treatment recommendation for lipoprotein (a) lowering therapy, for triglyceride lowering therapy, for cholesterol lowering therapy, and / or other dyslipidemia therapy.

7. The method according to any one of claims 1 - 6, wherein the inflammatory leukocytes and / or atherosclerotic plaque inflammation, atherosclerosis, and / or a cardiovascular disease is / are mediated at least partially by an increase in circulating foamy monocytes in the subject, and / or by an accumulation of foamy monocytes in atherosclerotic plaques in the subj ect .

8. The method according to any one of claims 1 - 7, wherein the cardiovascular disease comprises or is at least one of coronary artery disease, such as angina or myocardial infarction, stroke, heart attack, cerebrovascular disease, or peripheral artery disease .

9. The method according to any one of claims 1 - 8, wherein the subj ect does not exhibit hypercholesterolemia .

10. The method according to any one of claims 1 - 9, wherein the determining the quantitative value of the lipid droplets comprises determining at least one of the amount or relative amount, area, intensity or the number of lipid droplets in thecells, or the number or proportion of cells comprising lipid droplets .

11. The method according to any one of claims 1 - 10, wherein the quantitative value of lipid droplets is determined in naive cells; in cells incubated in a lipid-rich medium; and / or in cells incubated in a lipid-poor medium.

12. The method according to any one of claims 1 - 11, wherein the biological sample is a blood sample or a sample derived from a blood sample .

13. The method according to any one of claims 1 - 12, wherein the cells are leukocytes, such as peripheral blood mononuclear cells (PBMCs) , for example monocytes .

14. The method according to any one of claims 1 - 13, wherein the biological sample and / or the cells are frozen and / or processed at least 3 hours after the biological sample has been collected .

15. The method according to any one of claims 1 - 14, wherein the quantitative value of lipid droplets is determined using a semi-automated analysis pipeline .

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