New pathological markers and their uses

Abstract

The present invention relates to the identification of new pathological markers, a new method for diagnosing or monitoring the progression of non-alcoholic hepatic steatosis (NAFLD) by detecting and quantifying said markers, and a device capable of implementing said method.

Description

Technical Field

[0001] The present invention relates to the identification of new pathological markers, a new method for diagnosing or monitoring the progression of non-alcoholic hepatic steatosis NAFLD and / or NASH by detecting and quantifying said markers, and a device capable of implementing said method. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a group of liver histological changes characterized by excessive accumulation of fat within hepatocytes (steatosis, including >5% cellular triglyceride accumulation) in the absence of heavy alcohol consumption and secondary causes of liver disease.

[0003] NAFLD is currently the leading cause of altered hepatocellular carcinoma in adults in the Western world and, worryingly, in children and adolescents as well. Updated data reported in Bugianesi and Marietti 2016 (RecentiProg Med 2016;107:360-368) indicate that the global prevalence of NAFLD in the adult population is as high as 25%, and that the global prevalence of NASH (non-alcoholic hepatic steatosis) diagnosed by liver biopsy in individuals with NAFLD ranges from 20% to 50%. The percentages reported here rise sharply in patients at risk, such as those who are obese and / or have type 2 diabetes.

[0004] The most striking epidemiological data concern the pediatric population, where obesity and metabolic syndrome are increasing worldwide and are now evident in Europe, where estimates are similar to those recorded in the United States, with data recorded from 2007 to 2010 finding a prevalence of NAFLD of approximately 7%, three times the value recorded less than 20 years earlier.

[0005] While simple steatosis carries a negligible risk of progression to cirrhosis, a significant proportion (10-15%) of NAFLD subjects exhibit histological aspects of necroinflammation and ballooning, hallmarks of the most severe form of liver disease, nonalcoholic steatohepatitis (NASH), which can progress to fibrosis, cirrhosis, and associated complications, including hepatocellular carcinoma (HCC).

[0006] As a complex multifactorial pathology, the severity and onset of nonalcoholic hepatic steatosis are influenced by genetic and environmental factors. It is often shown to be associated with pathologies such as metabolic syndrome and type 2 diabetes, but it also plays a role in the development and progression of the syndrome and its associated complications. Given that the prevalence of NAFLD is increasing in affluent countries, which is associated with dietary and social changes, and because this pathology has potentially significant clinical implications, for example, in a non-negligible number of cases it may also evolve into nonalcoholic hepatic steatosis (a cause of cirrhosis and HCC), it is obviously important to be able to identify high-risk subjects early and correctly so that the development of complications that may affect prognosis can be investigated and predicted at the hepatic and extrahepatic (cardiovascular) levels.

[0007] In fact, in addition to liver damage, the major cause of morbidity and mortality in individuals with NAFLD is the cardiovascular complications that occur in these individuals more frequently than in the general population, regardless of the presence of type 2 diabetes and other risk factors.

[0008] As reported by Bugianesi and Marietti in 2016, since the diagnosis of NASH is currently conditional on liver biopsy, scientific efforts in recent years have been aimed at finding non-invasive markers of liver damage and related gene polymorphisms that can be applied on a large scale in order to properly address screening programs, follow-up actions, and treatment attempts.

[0009] The identification of noninvasive diagnostic tools could enable timely diagnosis even in subjects who would not normally undergo a liver biopsy, as biopsy of this organ is typically performed only when severe symptoms make it essential. Furthermore, noninvasive diagnostics would allow for continuous monitoring of the effectiveness of therapeutic treatments administered to patients. It should also be considered that ultrasound-guided liver biopsy is an expensive procedure that must be performed in a hospital setting and can cost anywhere from $2,000 to $7,000 or more. The mortality rate for fatal bleeding after liver biopsy ranges from 0.13% to 0.33% but may be higher in subjects with altered coagulation profiles.

[0010] Therefore, there is a great need to identify diagnostic markers for NAFLD that allow for accurate and rapid diagnosis without the need for liver biopsy intervention, thereby expanding the diagnosis of the disease in the population due to the non-invasive nature of the diagnostic method. Summary of the Invention

[0011] The authors of the present invention have found that the Plin2 protein is an effective marker for non-alcoholic hepatic steatosis (NAFLD) and non-alcoholic steatohepatitis (NASH) in circulating cells. Surprisingly, the expression level of the marker measured in hepatocytes is strongly positively correlated with the expression level measured in leukocytes, particularly in peripheral blood polymorphonuclear cells and monocytes of the same individual. In addition, the authors of the present invention also found that the expression level of the Plin2 protein in blood cells and hepatocytes is proportional to the NAS score of NAFLD and the severity of NASH, so that the higher the Plin2 concentration in patients with NAFLD, the higher the NAS score, and the higher the Plin2 concentration in patients with NASH, the greater the severity of the disease (NAS score and NASH severity are defined according to the literature and this specification).

[0012] Therefore, the authors of the present invention have designed a method for diagnosing NAFLD and / or NASH on a blood sample of an individual or on leukocytes extracted from said sample, wherein the diagnosis of NAFLD and / or NASH is considered to be confirmed if the concentration of Plin2 in said sample is greater than the concentration measured in one or more control samples taken from healthy individuals.

[0013] By virtue of the correlation between said expression and the severity of the disease, the measurement of the degree of Plin2 expression can also be advantageously used to monitor the effectiveness of treatment, whereby a comparison of the measurement of the Plin2 blood concentration of an individual, carried out at the moment t0 at which monitoring of the effectiveness of treatment begins, allows the evaluation of the effectiveness of said treatment by comparing the value obtained in said measurement with the value obtained in one or more subsequent measurements carried out at moments tn, where n is an integer greater than 0 and where said moments are progressively later (after or subsequent) than one another and are all after t0, since a decrease in the Plin2 concentration over time indicates the effectiveness of treatment. A constant value over time indicates that the disease is under control, while an increase in said value over time indicates that the treatment is ineffective.

[0014] Furthermore, measurements at later times as described above also enable monitoring of disease progression in the absence of pharmacological treatment, for example following changes in the patient's diet and lifestyle habits, medications or bariatric surgery.

[0015] Thus, the present invention advantageously enables diagnosis and / or monitoring of NAFLD or NASH without the need for a liver biopsy, which has clear and obvious medical and economic advantages. Furthermore, the simplicity of diagnosis and / or monitoring according to the present invention significantly expands the number of individuals who can be analyzed, allowing for easy testing of children and, furthermore, the ability to assess the health status of entire patient populations who would not normally undergo a liver biopsy for NAFLD or NASH. Furthermore, the analysis according to the present invention also enables population screening.

[0016] Furthermore, given the higher frequency of cardiovascular damage in patients with NAFLD and NASH, which has been shown to be independent of the presence or absence of other risk factors, early diagnosis of the disease would allow for preventive monitoring of the cardiovascular system in these patients.

[0017] The authors of the present invention have also found that the Plin2 protein concentration values ​​as described above are correlated with the NAS score and also with the severity grades of NASH, which are defined in the literature as mild, moderate and severe based on histological parameters observed in biopsy samples. Therefore, the present invention also relates to methods, computer programs and devices for defining the severity grade of NASH in patients with the pathology defined herein based on the Plin2 protein concentration values ​​in biological samples of said patients.

[0018] The authors of the present invention have also surprisingly found that the values ​​of Pnpla3 and Rab14 protein expression in the same biological sample can be used to diagnose the presence and severity of liver fibrosis.

[0019] The method of the invention can also be performed with simple devices capable of carrying out the above-mentioned measurements (determinations) and optionally processing the data obtained.

[0020] Therefore, the objects of the present invention are:

[0021] A method for diagnosing non-alcoholic hepatic steatosis (NAFLD) and / or non-alcoholic steatohepatitis (NASH), comprising the steps of:

[0022] a. measuring the concentration of Plin2 protein in a blood sample of an individual or in a leukocyte sample extracted from the blood sample,

[0023] b. comparing the value obtained at point a with the concentration value of Plin2 protein in a blood sample of a healthy individual or in a leukocyte sample extracted from the blood sample,

[0024] c. When the value measured in a is greater than the value measured in b, NAFLD and / or NASH is diagnosed;

[0025] A method for diagnosing NAFLD or NASH, comprising the steps of:

[0026] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0027] b. measuring the concentration values ​​of the Plin2 protein in a population of blood samples from patients with NAFLD and healthy patients, and identifying a cutoff value C0 of the concentration between patients with NAFLD and healthy patients, and measuring the concentration values ​​of the Plin2 protein in a population of blood samples from patients with NASH and healthy patients, and identifying a cutoff value C1 of the concentration between patients with NASH and healthy patients,

[0028] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0029] d. When the value obtained in a is greater than or equal to the value C0 and less than the value C1, it is diagnosed as NAFLD, or when the value obtained in a is greater than or equal to the value C1, it is diagnosed as NASH;

[0030] A method for diagnosing NAS in a patient with NAFLD or NASH, comprising the steps of:

[0031] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0032] b. measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NAFLD or NASH and healthy patients, wherein, in the population comprising samples from patients with NAFLD, NAS values ​​are histologically defined as NAS1, NAS2, and NAS3 in the patients based on the percentage (percentage) of steatosis in hepatocytes, the occurrence of balloon-like hepatocytes, the presence of lobular inflammation, and the presence of portal inflammation, and identifying:

[0033] cutoff value C2 of said concentration between healthy patients and patients with NAFLD of NAS1;

[0034] a cutoff value C3 of said concentration between patients with NAFLD of NAS1 and patients with NAFLD of NAS2; and

[0035] a cutoff value C4 of said concentration between patients with NAFLD of NAS2 and patients with NASH of NAS 3;

[0036] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0037] d. The NAS level is diagnosed as:

[0038] When the value obtained in a is greater than or equal to the cutoff value C2 and less than or equal to the cutoff value C3, NAS1,

[0039] When the value obtained in a is greater than the value C2 and less than or equal to the cutoff value C4, NAS2,

[0040] When the value obtained in a is greater than the value C4, NAS 3;

[0041] A method for diagnosing the severity level of NASH, comprising the steps of:

[0042] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0043] b. measuring the concentration value of Plin2 protein in a population of blood samples from patients with NASH and healthy patients, wherein, in the population comprising samples from patients with NASH, the severity grade of pathology is histologically defined as mild, moderate, or severe in the patients based on the percentage of steatosis in hepatocytes, the occurrence of balloon-like hepatocytes, the presence of lobular inflammation, and the presence of portal inflammation, and identifying:

[0044] The cutoff value C5 for the concentration between healthy patients and patients with mild NASH;

[0045] a cutoff value C6 for said concentration between patients with mild NASH and patients with moderate NASH; and

[0046] The cutoff value C7 of the concentration between patients with moderate NASH and patients with severe NASH,

[0047] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0048] d. Diagnose the severity of NASH as

[0049] When the value obtained in a is greater than the value C5 and less than the value C6, mild,

[0050] When the value obtained in a is greater than the value C6 and less than the value C7, moderate, and

[0051] When the value obtained in a is greater than the value C7, severe;

[0052] A method for diagnosing NAFLD or NASH, comprising the steps of:

[0053] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0054] b. comparing the value obtained in point a with a cutoff value of said concentration expressed as mean fluorescence intensity (MFI) equal to 2.7 MFI and a cutoff value of said concentration equal to 1.0 MFI,

[0055] d. NAFLD is diagnosed when the value obtained in a is greater than or equal to 1.0 MFI and less than or equal to 2.7 MFI, or NASH is diagnosed when the value obtained in a is greater than 2.7 MFI;

[0056] A method for diagnosing NAS in a patient with NAFLD or NASH, comprising the steps of:

[0057] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0058] c. comparing the value obtained in point a with the cutoff values ​​of said concentration expressed as mean fluorescence intensity (MFI) equal to 1.0 MFI, 1.4 MFI and 2.7 MFI,

[0059] d. When the value obtained in a is greater than or equal to the cutoff value of 1.0 MFI and less than or equal to the cutoff value of 1.4 MFI, it is diagnosed as NAS equal to 1; when the value obtained in a is greater than the cutoff value of 1.4 MFI, it is diagnosed as NAS equal to 2; or when the value obtained in a is greater than the cutoff value of 2.7 MFI, it is diagnosed as NAS equal to 3;

[0060] A method for diagnosing the severity level of NASH, comprising the steps of:

[0061] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0062] c. comparing the value obtained in point a with the cutoff values ​​of said concentration expressed as mean fluorescence intensity (MFI) equal to 2.7 MFI, 4 MFI and 6.3 MFI,

[0063] d. When the value obtained in a is greater than the cutoff value of 2.7 MFI and less than or equal to the cutoff value of 4 MFI, a mild form of NASH is diagnosed, when the value obtained in a is greater than the cutoff value of 4 MFI and less than or equal to the value of 6.3 MFI, a moderate form of NASH is diagnosed, and when the value obtained in a is greater than 6.3 MFI, a severe form of NASH is diagnosed;

[0064] The method further comprises the steps of: measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample or a leukocyte sample extracted from the blood sample at point a for diagnosing NASH, and comparing the concentration values ​​thereof with those in blood samples from healthy patients and optionally from patients with a histologically defined liver fibrosis grade, for the diagnosis of liver fibrosis;

[0065] A method for monitoring the effectiveness of therapeutic treatment of NAFLD or NASH in a patient, comprising the steps of:

[0066] a. measuring the concentration of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a leukocyte sample extracted from the blood sample at the start of the monitoring at time t0,

[0067] b. measuring the concentration of Plin2 protein in a blood sample of an individual with NAFLD or NASH or in a white blood cell sample extracted from the blood sample at one or more times tn, wherein n is an integer greater than 0, and wherein each tn corresponds to a time after t0, wherein

[0068] A decrease in the concentration of Plin2 protein at one or more of said moments tn indicates effectiveness of said therapeutic treatment;

[0069] A method for monitoring the progression of NAFLD or NASH in a patient, comprising the steps of:

[0070] a. measuring the concentration of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a leukocyte sample extracted from the blood sample at the start of the monitoring at time t0,

[0071] b. measuring the concentration of Plin2 protein in a blood sample of an individual with NAFLD or NASH or in a white blood cell sample extracted from the blood sample at one or more times tn, wherein n is an integer greater than 0, and wherein each tn corresponds to a time after t0, wherein

[0072] A decrease in the Plin2 protein concentration at one or more of the time moments tn indicates an improvement in NAFLD or NASH, while an increase in the Plin2 protein concentration at one or more of the time moments tn indicates a worsening of NAFLD or NASH;

[0073] A computer program for easily implementing the method;

[0074] And a device for automatically measuring the concentration value of Plin2 protein in a blood sample, generally comprising:

[0075] Device for isolating PBMCs from blood samples;

[0076] a device for measuring the concentration of Plin2 protein in the cytoplasm of the PBMC, and

[0077] A control unit connected to the separating device and the measuring device, which is programmed in the following manner:

[0078] controlling and synchronizing their drives according to automatic mode according to predetermined operating parameters, and

[0079] The data of the Plin2 protein concentration in the cytoplasm of the PBMCs were automatically compared with the reference data of the Plin2 protein concentration.

[0080] Glossary

[0081] The term leukocyte for the purposes of the present invention has its well-known meaning in the literature and includes different cell types: granulocytes (or polymorphonuclear cells), which are subdivided into neutrophils, eosinophils or acidophils, basophils; lymphocytes; monocytes.

[0082] For the purposes of the present invention, the term Plin2 denotes the human protein adipose differentiation-related protein, also known as ADFP, ADRP or lipid droplet coating protein 2, which is encoded by the human gene ADFP located at 9p22.1.

[0083] NAFLD Non-alcoholic hepatic steatosis

[0084] NASH nonalcoholic steatohepatitis

[0085] Pnpla3 refers to human Pnpla3 protein, also known as adiponutrin, ADPN, acylglycerol O-acyltransferase, C22orf20, IPLA2epsilon, DJ796117.1, IPLA(2)epsilon, and IPLA2-epsilon.

[0086] Rab14 refers to the human Rab14 protein, also known as the Ras-related protein Rab-14. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Panel A: Lipid droplets (light gray dots) in monocytes and hepatocytes: Large, weakly stained nuclei are visible. Panel B shows a Western blot of Plin2 in monocytes and liver. β-actin shows that the amount of protein detected in monocytes is similar to that detected in hepatocytes.

[0088] Figure 2 reported ORO staining in liver sections and mononuclear cells from the same subjects.

[0089] Figure 3 Report the Bland Altman diagram of the difference (Y axis) and mean value (X axis) of Plin2 measured by Western blot in monocytes and liver. Two dotted lines represent confidence limits. The difference between the values ​​obtained by the two measurements is reported on the Y axis, and the mean value is reported on the Y axis. The dotted line reports the 95% confidence limits (limits) between the two measurements. The figure shows that all points are within the confidence limits, which indicates that there is a direct correlation between Plin2 expression in the liver and peripheral blood cells.

[0090] Figure 4 : An exemplary block diagram of a preferred embodiment of the device according to the present invention.

[0091] Figure 5 : An example diagram of use of another preferred embodiment of the device according to the present invention.

[0092] Figure 6 : An example diagram of use of another preferred embodiment of the device according to the present invention.

[0093] Figure 7 : Correlation between mean Plin2 (MFI) level and NAS stage, the independent variable on the X-axis is the Plin2 adjustment value, and the dependent variable is the NAS stage.

[0094] Figure 8 : Importance of Plin2 normalization in NASH.

[0095] Figure 9 : Importance of Pnpla3 and Rab14 for fibrosis normalization.

[0096] Figure 10 : Importance of Plin2 for fibrosis normalization. DETAILED DESCRIPTION

[0097] The authors of the present invention were surprised to find that the Plin2 protein is a marker for NAFLD and NASH, that the protein concentration in blood cells (leukocytes) is correlated with the expression of the protein in liver cells, and that its concentration is proportional to the NAS score and also to the severity grade of the NASH disease. Therefore, the authors of the present invention have found that by analyzing blood samples without resorting to a liver biopsy, the measurement of the Plin2 protein concentration can be used to diagnose NAFLD or NASH, diagnose its severity grade as mild, moderate or severe as defined in the literature, or monitor the effectiveness of therapeutic treatments for NAFLD or NASH, or also monitor the progression of NAFLD or NASH over time.

[0098] Therefore, the present invention provides a method for diagnosing non-alcoholic hepatic steatosis (NAFLD) and / or non-alcoholic steatohepatitis (NASH), comprising the following steps:

[0099] a. measuring the concentration of Plin2 protein in a blood sample of an individual or in a leukocyte sample extracted from the blood sample,

[0100] b. comparing the value obtained at point a with the concentration value of Plin2 protein in a blood sample of a healthy individual or in a leukocyte sample extracted from the blood sample,

[0101] c. When the value measured in a is greater than the value measured in b, NAFLD and / or NASH is diagnosed.

[0102] In one embodiment, a blood sample is used in step a, which can be processed by techniques known to those skilled in the art to separate leukocytes therefrom.

[0103] Therefore, the method may include, before measuring the Plin2 protein concentration in the sample, treating the blood sample to separate the leukocytes therein. In one embodiment, the method may include separating polymorphonuclear cells and / or monocytes from the blood sample to be analyzed or from the leukocytes separated therefrom.

[0104] Any protocol currently known in the literature for isolating leukocytes from a blood sample may be used.

[0105] By way of example only, polymorphonuclear cells can be isolated from a blood sample as follows: the blood is collected into a tube containing EDTA (final concentration 4 mM) to prevent it from clotting. Then, one or more discontinuous Percoll gradients (composed of colloidal silica particles with a diameter of 15-30 nm that have been coated with polyvinylpyrrolidone (PVR) (23% w / w in water)) are set up, 15 ml of 62% Percoll is placed in a tube, and with the help of a syringe with a fine cannula, 15 ml of 75% Percoll is gently layered underneath it, avoiding mixing of the two suspensions.

[0106] 8-10 ml of blood was then layered onto the gradient; the tubes were centrifuged at 20°C for a total of 25 minutes, including 10 minutes at 200 rpm and then 15 minutes at 400 rpm. Thus, the blood components were separated based on density and size: erythrocytes and most eosinophils sedimented at the bottom of the tube; polymorphonuclear cells sedimented at the interface between the two Percoll suspensions; and lymphocytes were located between the 62% Percoll and plasma.

[0107] After discarding the plasma and lymphocytes, the band containing polymorphonuclear cells was collected using a glass Pasteur pipette, collected in a test tube, and washed in HEPES / BSA by centrifugation at 250 rpm for 7 minutes at 20°C. To discard any red blood cells, the bottom layer thus obtained was rapidly lysed and resuspended in 3 parts of hypotonic solution. After stirring for approximately 15 seconds, the medium was restored to isotonicity by adding 7 parts of isotonic solution.

[0108] After a further wash at 250 rpm for 7 minutes at 20°C, the cell pellet was resuspended and kept in a known volume of HEPES / BSA until use. Neutrophil concentration was determined using an electronic particle counter or a hemocytometer under a light microscope.

[0109] By way of example only, various mononuclear cell types can be isolated from blood samples by the following technique: CD-specific recombinant Fab fragments expressed by microorganisms using monoclonal antibodies against surface cell markers. In addition, commercial kits are available for this purpose, e.g., FABian TM IBA GmbH TM CD14 Isolation Kit.

[0110] The measurement of Plin2 protein concentration can be carried out according to any method available to those skilled in the art, and as an example only, in the methods described herein, measurement can be by western blotting, or cytofluorimetry, or ELISA, or immunofluorescence, or quantitative PCR, enzyme-linked immunosorbent assay (ELISA), or localized surface plasmon resonance fiber tip probe system, or by using the equipment prepared for the measurement described below. All of the above methods are known to those skilled in the art, and they know which sample needs to be analyzed and which protein should be quantified, and they can choose the most suitable scheme and use the most suitable reagents on the market. All reagents required for Plin2 detection and quantification in the sample according to this specification are known and available on the market, so once knowing the method of the present invention, those skilled in the art do not need special inventions.

[0111] By way of example, the techniques listed above are summarized and steps commonly used by those skilled in the art for each of them are indicated.

[0112] Western blotting monitors protein expression in cells to determine the presence, amount, and molecular weight of a specific antigen through three processes:

[0113] 1) Protein extraction and dosage;

[0114] 2) Protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); 3) Western blotting: Proteins separated from the gel are transferred to nitrocellulose for analysis (blotting); the membrane is exposed to an antibody against the target protein (primary antibody); the membrane is exposed to an antibody against the antibody substance used as the primary antibody (secondary antibody), and the membrane is developed using a chemiluminescent method (ECL).

[0115] 4) Image density analysis.

[0116] Cytofluorimetry is a laboratory technique that allows the detection, identification, and counting of specific cells or the proteins contained therein.

[0117] The cytofluorimetry technique envisages multiple stages:

[0118] The cell sample is suspended in a fluid.

[0119] Before the test, and depending on the cells to be analyzed, the sample is treated with a specific dye that can distinguish between cell subtypes. This dye (fluorochrome) is combined with a monoclonal antibody directed against a specific cell region or marker antigen.

[0120] The sample containing the labeled cells is introduced into an instrument called a cytofluorimeter.

[0121] In this instrument, a fluid containing cells is directed into a flow chamber and then through a very narrow orifice, creating a stream in which the cells are organized into rows one after another. The stream of cells is placed in front of a detector, which analyzes each cell present in the stream at a very high rate (hundreds to thousands of cells per second).

[0122] The cytofluorimeter contains one or more lasers and multiple detectors that can identify some characteristics unique to each cell. Each laser strikes the cells present in the flow, producing a characteristic scattering for each of them according to its characteristics. These characteristics can be physical (cell size and complexity) or can depend on the signal generated by the laser intercepting dye (fluorescent dye). The combination of this information generates a characteristic spectrum for each cell present in the sample. The signals detected by multiple detectors (or one detector) are amplified (by photomultiplier tubes) and sent to a computer. Here, it is converted into a digital format and displayed on a computer or printed.

[0123] The data are provided in the form of graphs.

[0124] ELISA (enzyme-linked immunosorbent assay) is a widely used technique based on the chemical conjugation of enzymes to antibodies or antigens. The activity of these enzymes is easily monitored and allows for accurate quantification of the concentration of the conjugated complex. Depending on the specific method used, ELISA can be used to quantify either antigens or antibodies.

[0125] Antigens are recognized by specific antibodies (Immuno).

[0126] The analyte (antigen or antibody) is adsorbed on the surface of the system (adsorbent).

[0127] The antigen / antibody recognized by the (secondary) enzyme-linked antibody can produce a reaction whose product is stained.

[0128] Quantitative PCR or real-time PCR is a technique for quantifying nucleic acids (in this case, mRNA encoding the Plin2 protein) by measuring the fluorescence emitted by a fluorophore. This technique links amplification and quantification in a single reaction. In a real-time PCR reaction, fluorescence increases in proportion to the accumulation of PCR products. Those skilled in the art have no problem designing probes suitable for RT-PCR, as the gene and DNA encoding the Plin2 protein are known in the literature. The enzyme-linked immunosorbent spot (ELISPOT) analysis in PBMC samples is based on incubating cells for a defined period of time in 96-well plates that have been functionalized (coated) with the adsorption of a monoclonal antibody with high affinity for the cytokines under study produced during the incubation period. In the presence of an enzyme chromogenic substrate, a combination of an anti-reference protein biotinylated antibody and an avidinase-linked secondary antibody can detect proteins produced in a specific area due to precipitation of reaction products by the formation of spots (dye accumulation). The technique can be applied to PBMC content after cell lysis. Surface plasmons are used to improve the surface sensitivity of various spectroscopic measurements (including fluorescence, Raman scattering, and second harmonic generation). However, in their simplest form, SPR reflectance can be used to detect molecular absorption in proteins etc. Technically speaking, the angle of minimum reflection (maximum absorption) is measured.

[0129] Any analytical technique known in the literature that is suitable for quantifying the concentration of Plin2 in a blood or cell sample as defined above can be used in any method of the invention.

[0130] Furthermore, the device according to the present invention can be designed to quantify Plin2 protein in a target sample by one or more of the above-mentioned detection techniques.

[0131] In a specific embodiment, the measurement can be performed by cytofluorimetry using an antibody specific for Plin2 protein (ie, binding only to Plin2 protein) (or a derivative or fragment thereof as defined above) labeled with a suitable fluorescent dye as a protein marker.

[0132] In one embodiment, the fluorescent dye can be any fluorescent dye detectable by a commercially available cytofluorimeter, such as a fluorescent dye having the following characteristics:

[0133]

[0134] In one embodiment, Alexa Fluor 488 fluorescent dye (Invitrogen) can be used, which is commonly used as a substitute for FITC or Cy2 and has the following technical features.

[0135] Analysis of fluorescence intensity and mean fluorescence intensity (MFI) values ​​can be calculated by suitable software available to the public and evaluated by using a commercially available cytofluorimeter.

[0136] Therefore, according to one embodiment, the fluorescent dye used can be AlexaFluor 488, a cell fluorometer FC 500 (Beckman Coulter, Brea, CA) having the above-mentioned technical features, and the technical features of the product described at the time of filing this application can be analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0137] In any case, one skilled in the art will know how to adjust the cutoff values ​​provided herein expressed in MFI calculated using Alexa Fluor 488 fluorescent dye or fluorescent dyes having the characteristics reported in the above table using a cytofluorometer FC 500 (Beckman Coulter, Brea, CA) or a cytofluorometer with similar technical features and Kaluza software (Beckman Coulter, Brea, CA) by comparative analysis of the cutoff values ​​expressed in MFI calculated using other fluorescent dyes and other equipment and software.

[0138] The MFI is simply the arithmetic mean.

[0139] According to the present invention, the cytofluorometer may preferably have the technical features of the above-described cytofluorometer, as publicly available at the time of filing of the present application.

[0140] All embodiments of the above steps are applicable to any method provided in this specification.

[0141] In one embodiment, the present invention relates to a method for diagnosing NAFLD or NASH, comprising the following steps:

[0142] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0143] b. measuring the concentration values ​​of the Plin2 protein in a population of blood samples from patients with NAFLD and healthy patients, and identifying a cutoff value C0 of the concentration between patients with NAFLD and healthy patients, and measuring the concentration values ​​of the Plin2 protein in a population of blood samples from patients with NASH and healthy patients, and identifying a cutoff value C1 of the concentration between patients with NASH and healthy patients,

[0144] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0145] d. When the value obtained in a is greater than or equal to the value C0 and less than the value C1, NAFLD is diagnosed, or when the value obtained in a is greater than or equal to the value C1, NASH is diagnosed.

[0146] The present invention also relates to a method for diagnosing NAS score in a patient suffering from NAFLD or NASH, comprising the following steps:

[0147] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0148] b. measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NAFLD or NASH and healthy patients, wherein, in the population comprising samples from patients with NAFLD, NAS score values ​​are histologically defined as NAS1, NAS2, and NAS3 in the patients based on the percentage of steatosis in hepatocytes, the occurrence of balloon-like hepatocytes, the presence of lobular inflammation, and the presence of portal inflammation, and identifying:

[0149] cutoff value C2 of the concentration between healthy patients and patients with NAFLD with NAS score 1;

[0150] a cutoff value C3 of said concentration between patients with NAFLD of NAS 1 and patients with NAFLD of NAS score 2; and

[0151] The cutoff value C4 for the concentration between patients with NAFLD with NAS score 2 and patients with NASH with NAS score 3;

[0152] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0153] d. The NAS score is diagnosed as:

[0154] When the value obtained in a is greater than or equal to the cutoff value C2 and less than or equal to the cutoff value C3, the NAS score is 1,

[0155] When the value obtained in a is greater than the value C2 and less than or equal to the cutoff value C4, the NAS score is 2,

[0156] When the value obtained in a is greater than the value C4, the NAS score is 3.

[0157] The NAS score according to the present invention is as defined in the literature and is generally assigned according to the following parameters:

[0158]

[0159]

[0160] The present invention also relates to a method for diagnosing the severity level of NASH, comprising the following steps:

[0161] a. measuring the concentration of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample,

[0162] b. measuring the concentration value of Plin2 protein in a population of blood samples from patients with NASH and healthy patients, wherein, in the population comprising samples from patients with NASH, the severity grade of pathology is histologically defined as mild, moderate, or severe in the patients based on the percentage of steatosis in hepatocytes, the occurrence of balloon-like hepatocytes, the presence of lobular inflammation, and the presence of portal inflammation, and identifying:

[0163] The cutoff value C5 for the concentration between healthy patients and patients with mild NASH;

[0164] a cutoff value C6 for said concentration between patients with mild NASH and patients with moderate NASH; and

[0165] The cutoff value C7 of the concentration between patients with moderate NASH and patients with severe NASH,

[0166] c. Compare the value obtained in point a with the cutoff value obtained in point b,

[0167] d. Diagnose the severity of NASH as:

[0168] When the value obtained in a is greater than the value C5 and less than the value C6, mild,

[0169] When the value obtained in a is greater than the value C6 and less than the value C7, moderate, and

[0170] When the value obtained in a is greater than the value C7, it is serious.

[0171] Therefore, the cut-off value for Plin2 concentration according to the present specification is:

[0172] The cutoff value C0 between patients with NAFLD and healthy patients,

[0173] The cutoff value C1 between patients with NASH and healthy patients,

[0174] The cutoff value C2 between healthy patients and patients with NAFLD of NAS1,

[0175] The cutoff value C3 between patients with NAFLD of NAS1 and patients with NAFLD of NAS2,

[0176] The cutoff value C4 between patients with NAFLD at NAS2 and patients with NASH at NAS3,

[0177] The cutoff value C5 between healthy patients and patients with mild NASH,

[0178] The cutoff value C6 between patients with mild NASH and patients with moderate NASH,

[0179] The cutoff value C7 was used between patients with moderate NASH and patients with severe NASH.

[0180] The definition of disease severity according to the present invention is based on the definition commonly used in the literature based on histological analysis according to the NAFLD Activity Score (NAS) (Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. HEPATOLOGY 2005; 41: 1313-1321.). This is a widely used histological score validated by liver disease societies around the world, based on the presence of steatosis, lobular and portal inflammation, and ballooning hepatocyte degeneration ("ballooning") in liver biopsies; each component can be graded 1 (mild), 2 (moderate), and 3 (severe).

[0181]

[0182] In fact, the authors of the present invention surprisingly found that the concentration values ​​of Plin2 protein in analyzed blood samples from patients with known NASH severity grade correlate with said grade and that the increase in Plin2 protein concentration is proportional to the severity of this pathology.

[0183] In a preferred embodiment, the above method is performed by using cytofluorescence technology to assess Plin2 protein expression using a monoclonal antibody that specifically binds to Plin2 and does not bind to other proteins and is labeled with an appropriate fluorescent dye, and the cutoff value is expressed as mean fluorescence intensity (MFI).

[0184] In a more preferred embodiment, it is performed by using the above-mentioned fluorescent dyes, software and equipment, or also the above-mentioned fluorescent dyes and software and equipment that are the subject of the invention as defined below and in the claims. According to the literature and according to the invention, the NASH necroinflammation grade is divided into grade 1 (mild), grade 2 (moderate) and grade 3 (severe) according to the grade of hepatocyte steatosis, ballooning and disorganization and inflammation (intralobular and portal areas) (above table).

[0185] The authors of the present invention have demonstrated (see Examples section) that it is possible to calculate specific cut-off values ​​(expressed herein as MFI using fluorescent dyes, software and an instrument as defined below) suitable for the above method.

[0186] Therefore, the present invention also aims to:

[0187] A method for diagnosing NAFLD or NASH, comprising the following steps:

[0188] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0189] b. comparing the value obtained in point a with a cutoff value of said concentration expressed as mean fluorescence intensity (MFI) equal to 2.7 MFI and a cutoff value of said concentration equal to 1.0 MFI,

[0190] d. NAFLD was diagnosed when the value obtained in a was greater than or equal to 1.0 MFI and less than or equal to 2.7 MFI, or NASH was diagnosed when the value obtained in a was greater than 2.7 MFI.

[0191] A method for diagnosing NAS scoring in a patient with NAFLD or NASH, comprising the following steps:

[0192] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0193] c. comparing the value obtained in point a with the cutoff values ​​of said concentration expressed as mean fluorescence intensity (MFI) equal to 1.0 MFI, 1.4 MFI and 2.7 MFI,

[0194] d. A NAS score of 1 is diagnosed when the value obtained in a is greater than or equal to the cutoff value of 1.0 MFI and less than or equal to the cutoff value of 1.4 MFI; a NAS score of 2 is diagnosed when the value obtained in a is greater than the cutoff value of 1.4 MFI; or a NAS score of 3 is diagnosed when the value obtained in a is greater than the cutoff value of 2.7 MFI; and

[0195] A method for diagnosing the severity level of NASH, comprising the following steps:

[0196] a. measuring the concentration value of Plin2 protein in a blood sample or a leukocyte sample extracted from the blood sample, wherein the value is measured by cytofluorimetry using a monoclonal antibody that specifically binds Plin2 and does not bind to other proteins labeled with a suitable fluorescent dye,

[0197] c. comparing the value obtained in point a with the cutoff values ​​of said concentration expressed as mean fluorescence intensity (MFI) equal to 2.7 MFI, 4 MFI and 6.3 MFI,

[0198] d. When the value obtained in a is greater than the cutoff value of 2.7 MFI and less than or equal to the cutoff value of 4 MFI, a mild form of NASH is diagnosed, when the value obtained in a is greater than the cutoff value of 4 MFI and less than or equal to the value of 6.3 MFI, a moderate form of NASH is diagnosed, and when the value obtained in a is greater than 6.3 MFI, a severe form of NASH is diagnosed.

[0199] Such a method is to use Alexa Fluor 488 fluorescent dye, or a fluorescent dye having the characteristics reported in the above table, using an embodiment of the FC 500 cell fluorometer (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA). Similarly, different cutoff values ​​C0-C7 can be defined by using different detection and quantification systems of Plin2 protein.

[0200] As mentioned above, the authors also surprisingly found that the assessment of Pnpla3 and Rab14 protein concentrations in a blood sample or in a leukocyte sample extracted from said blood sample at point a of diagnosis of NASH can determine the presence or absence of liver fibrosis and its severity grade.

[0201] In the present invention, to define liver fibrosis, the same definition used in the literature (Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histologicalscoring system for nonalcoholic fatty liver disease. HEPATOLOGY 2005; 41: 1313-1321) is used, based on a scoring system for staging based on the location and extension of fibrosis: stage 1, area 3 perisinusoidal fibrosis; stage 2, portal fibrosis with the above stage 1; stage 3, bridging fibrosis in addition to stage 2; and stage 4, cirrhosis. The NASH Clinical Research Network (NASH CRN) subsequently subdivided stage 1 into 3 categories: stage 1A, mild perisinusoidal fibrosis in area 3; stage 1B, moderate perisinusoidal fibrosis in area 3; and stage 1C, only portal / periportal fibrosis. Stage 1C fibrosis is occasionally observed in children or severely obese patients.

[0202] Histological changes Fibrosis stage (SAFF) No fibrosis 0 Peri-sinusoidal or peri-portal fibrosis 1 Peri-sinusoidal or peri-portal fibrosis 2 Bridging fibrosis 3 Cirrhosis 4

[0203] Therefore, the present invention also provides a further step applicable to the above method, wherein, once NASH is diagnosed from a blood sample of a patient, the presence or absence of fibrosis and its pathological stage can also be diagnosed from the same sample or another blood sample of the patient. The step of fibrosis stage diagnosis can also be performed on a blood sample of a patient suffering from NASH as defined in the present specification and claims, wherein the diagnosis of NASH has been performed using a method different from that described and claimed in the present invention.

[0204] Therefore, in any embodiment described herein, step e may also be replaced by step e': measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample of a patient diagnosed with NASH or in a white blood cell sample extracted from a blood sample, and thus step g may be replaced by step g': comparing the value obtained at point e' with the cutoff value obtained at point f.

[0205] Therefore, the object of the present invention is any method for diagnosing single NASH and / or its severity, further comprising the following steps:

[0206] e. measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample or a leukocyte sample extracted from the blood sample in point a of diagnosing NASH,

[0207] f. measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a population of blood samples from patients with liver fibrosis and healthy patients, and identifying a cutoff value C8 of the concentration between patients with liver fibrosis and healthy patients,

[0208] g. Compare the value obtained in point e with the cutoff value obtained in point f,

[0209] h. when the value obtained at point e is greater than the value C8, liver fibrosis is diagnosed; or

[0210] A method for diagnosing liver fibrosis comprising steps e', f, g' and h.

[0211] The present invention also relates to any method for diagnosing single NASH and / or its severity, further comprising the following steps:

[0212] e. measuring the protein concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample or a leukocyte sample extracted from the blood sample in point a of diagnosing NASH,

[0213] f. Measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a population of blood samples from healthy patients and patients with liver fibrosis, wherein the severity grade of the pathology is histologically defined as stage 1, stage 2, or stage 3 based on the location and extension of liver fibrosis, and identifying:

[0214] The cutoff value C9 for the concentration between healthy patients and patients with stage 1 liver fibrosis;

[0215] a cutoff value C10 for said concentration between patients with stage 1 liver fibrosis and patients with stage 2 liver fibrosis; and

[0216] The cut-off value C11 of the concentration between patients with stage 2 liver fibrosis and patients with stage 3 liver fibrosis,

[0217] g. Compare the value obtained in point e with the cutoff value obtained in point f,

[0218] h. Diagnose the stage of liver fibrosis as:

[0219] When the value obtained at point e is greater than or equal to the value C9 and less than the value C10, stage 1,

[0220] When the value obtained at point e is greater than or equal to the value C10 and less than or equal to the value C11, stage 2, and

[0221] When the value obtained in point e is greater than said value C11, stage 3; or

[0222] A method for diagnosing the stage of liver fibrosis comprising steps e', f, g' and h', wherein all references to point e of the above step h are changed to references to point e', with necessary modifications.

[0223] The present invention also relates to a method for diagnosing liver fibrosis on a blood sample or lymphocytes from a patient diagnosed with NASH and a method for diagnosing the stage of liver fibrosis, comprising the above steps, wherein steps e and g are replaced by steps e' and g'.

[0224] In summary, the cutoff values ​​for Pnpla3 and Rab14 concentrations defined in this paper are:

[0225] The cutoff value C8 between patients with liver fibrosis and healthy patients,

[0226] The cutoff value C9 between healthy patients and patients with stage 1 liver fibrosis,

[0227] The cutoff value C10 between patients with stage 1 fibrosis and those with stage 2 fibrosis,

[0228] The cutoff value between patients with stage 1 liver fibrosis and those with stage 3 liver fibrosis was C11.

[0229] The concentration of the above proteins can be measured by Western blotting, cytofluorimetry, ELISA, or quantitative PCR.

[0230] In a specific embodiment, it can be measured by cytofluorimetry using a monoclonal antibody that specifically binds to Pnpla3 and does not bind to other proteins and a monoclonal antibody that specifically binds to Rab14 and does not bind to other proteins, which are labeled with appropriate fluorescent dyes, and the cutoff value is expressed as mean fluorescence intensity (MFI).

[0231] As described above, the concentration values ​​of Pnpla3 and Rab14 proteins can be calculated by using all the embodiments and devices described for calculating the concentration value of Plin2, such as fluorescent dyes having the following characteristics:

[0232]

[0233] Therefore, according to one embodiment, the fluorescent dye used can be AlexaFluor 488, a cell fluorometer FC 500 (Beckman Coulter, Brea, CA) having the above-mentioned technical features, and the technical features of the product available to the public at the time of filing this application can be analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0234] The authors of the present invention have demonstrated (see Examples section) that it is possible to calculate specific cut-off values ​​(expressed herein as MFI using fluorescent dyes, software and an instrument as defined below) suitable for the above method.

[0235] Therefore, the present invention also relates to any method for diagnosing single NASH and / or its severity, further comprising the following steps:

[0236] e. measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample or a leukocyte sample extracted from the blood sample in point a for diagnosing NASH, wherein the values ​​are measured by cytofluorimetry using a monoclonal antibody that specifically binds to Pnpla3 and does not bind to other proteins and a monoclonal antibody that specifically binds to Rab14 and does not bind to other proteins, which are labeled with a suitable fluorescent dye,

[0237] g. comparing the value obtained in point e with a cutoff value expressed as mean fluorescence intensity (MFI) greater than or equal to 1.24 MFI,

[0238] h. When the value obtained in point e is greater than or equal to 1.24 MFI, liver fibrosis is diagnosed.

[0239] and any method for diagnosing single NASH and / or its severity, further comprising the steps of:

[0240] e. measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample or a leukocyte sample extracted from the blood sample in point a for diagnosing NASH, wherein the values ​​are measured by cytofluorimetry using a monoclonal antibody that specifically binds to Pnpla3 and does not bind to other proteins and a monoclonal antibody that specifically binds to Rab14 and does not bind to other proteins, which are labeled with a suitable fluorescent dye, and the cutoff value is expressed as mean fluorescence intensity (MFI).

[0241] g. comparing the value obtained in point a with the cutoff values ​​of said concentration expressed as mean fluorescence intensity (MFI) equal to 1.24 MFI, 2.3 MFI and 3.10 MFI,

[0242] h. When the value obtained in point e is greater than or equal to 1.24 MFI and less than 2.4 MFI, it is diagnosed as mild stage 1 liver fibrosis, when the value obtained in point e is greater than or equal to 2.4 MFI and less than 3.10 MFI, it is diagnosed as stage 2 liver fibrosis, and when the value obtained in point e is greater than or equal to 3.10 MFI, it is diagnosed as stage 3 liver fibrosis.

[0243] Also in this context, the present invention relates to a method for diagnosing liver fibrosis on a blood sample or lymphocytes from a patient diagnosed with NASH and a method for diagnosing the stage of liver fibrosis, comprising the above-mentioned steps, wherein steps e and g are replaced by steps e', g' and h', and wherein all references to point e of the above-mentioned step h are changed to references to point e', mutatis mutandis.

[0244] The above reported values ​​are defined using Alexa Fluor 488 fluorescent dye or fluorescent dyes having the characteristics reported in the above table using a cytofluorimeter FC 500 (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA). Obviously, other cutoff values ​​can be specified by using different detection systems.

[0245] In any case, one skilled in the art will know how to adjust the cutoff values ​​provided herein expressed as MFI calculated using the Alexa Fluor 488 fluorescent dye or a fluorescent dye having the characteristics reported in the above table using a cytofluorimeter FC 500 (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA) by comparative analysis of cutoff values ​​calculated using other fluorescent dyes and other equipment and software.

[0246] Furthermore, the present invention provides a method for monitoring the effectiveness of a therapeutic treatment of NAFLD or NASH in a patient, comprising the steps of:

[0247] a. measuring the concentration of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a leukocyte sample extracted from the blood sample at the start of the monitoring at time t0,

[0248] b. measuring the concentration of Plin2 protein in a blood sample of an individual with NAFLD or NASH or in a white blood cell sample extracted from the blood sample at one or more times tn, wherein n is an integer greater than 0, and wherein each tn corresponds to a time after t0, wherein

[0249] A decrease in the concentration of Plin2 protein at one or more of said moments tn is indicative of the effectiveness of said therapeutic treatment.

[0250] In the execution of the above-described monitoring method, time t0 is the time at which monitoring starts, which may correspond to the moment before treatment starts, or may be the moment at which monitoring starts even if treatment has already started.

[0251] The times tn are times that follow (subsequently) each other as the value of n increases, and are after the time t0.

[0252] Therefore, time t0 is the time to start monitoring, and the concentration value of Plin2 in the blood sample is considered to be the value at the start of evaluation of treatment effectiveness. Then, after t0 and at times following each other (following) in the progression from 1 to time tn, the concentration measurement is repeated, where n is an integer greater than 0, where time t1 is before time t2, time t2 is before time t3, and so on.

[0253] A detectable, significant decrease in the concentration of Plin2 in a patient's blood sample analyzed at a time after t0 relative to the value measured at t0 indicates the effectiveness of the therapeutic treatment in improving NAFLD or NASH. A value that remains constant over time indicates the effectiveness of the therapeutic treatment in preventing the progression of NAFLD or NASH, and an increase in the value over time relative to the value measured at t0 indicates that the therapeutic treatment is ineffective.

[0254] In some cases, patients may be advised to make dietary and lifestyle changes before drug treatment is initiated. In these cases, it may be useful to monitor the progression of the disease over time to see whether, for example, dietary and lifestyle changes have a positive effect on the disease.

[0255] Regardless of whether or not the effectiveness of a possible treatment is being assessed, monitoring the course of the disease may be of interest to the treating physician. Therefore, the present invention is also directed to a method for monitoring the progression of NAFLD or NASH in a patient over time, comprising the following steps:

[0256] a. measuring the concentration of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a leukocyte sample extracted from the blood sample at the start of the monitoring at time t0,

[0257] b. measuring the concentration of Plin2 protein in a blood sample of an individual with NAFLD or NASH or in a white blood cell sample extracted from the blood sample at one or more times tn, wherein n is an integer greater than 0, and wherein each tn corresponds to a time after t0, wherein

[0258] A decrease in Plin2 protein concentration at one or more of said moments tn indicates an improvement in NAFLD or NASH, whereas an increase in Plin2 protein concentration at one or more of said moments tn indicates a worsening of NAFLD or NASH, whereas a constant value over time indicates a case of disease stagnation.

[0259] The description of the times t0 and tn in the explanation of the method for monitoring a therapeutic treatment applies to the general monitoring method.

[0260] All embodiments described with respect to the diagnostic method also apply to the monitoring method described below.

[0261] Generally, peripheral blood is preferably used when performing the methods described herein, although blood samples can also be collected from organs and the like.

[0262] In particular, in the implementation of the method described herein, it is preferred to measure the concentrations of Plin2, Pnpla3, and Rab14 in leukocytes, more specifically polymorphonuclear cells and / or monocytes, separated from the peripheral blood.

[0263] When using Pnpla3 and Rab14 concentration values ​​in blood samples of patients with hepatic steatosis as markers to monitor the effectiveness or progression of treatment of hepatic steatosis, the same methods described above for monitoring effectiveness and disease progression for NAFLD or NASH can be performed with necessary modifications.

[0264] The present invention also provides a device for automatically measuring the concentration of Plin2 protein and / or Pnpla3 and / or Rab14 in a blood sample collected from an individual, generally comprising:

[0265] Device for isolating PBMCs from blood samples;

[0266] a device for measuring the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins in the cytoplasm of the PBMC, and

[0267] Means for correlating the obtained measured values ​​with comparative values, which may be values ​​obtained from a healthy individual and / or previously obtained values ​​of one or more concentrations of Plin2 and / or Pnpla3 and / or Rabl4 proteins in the cytoplasm of PBMCs from a blood sample of the same individual.

[0268] As described above, the device according to the present invention may be implemented to measure Plin2 and / or Pnpla3 and / or Rab14 proteins in a blood sample taken from an individual using one or more of the above-described detection techniques.

[0269] Depending on the technique chosen for PBMC isolation and lysis, the above-described apparatus may be implemented according to various variants, some of which are described below. For example, in the case of using a separation format that selectively binds to components of PBMCs or monocytes, the apparatus may include:

[0270] - a collection chamber adapted to receive a blood sample;

[0271] - a mixing device included in the collection chamber, configured to mix the blood sample with a component susceptible to binding to polymorphonuclear cells (PBMCs) present in the sample and, if necessary, with an anticoagulant substance;

[0272] - a moving device configured to apply mechanical vibration and / or rotational stress to the mixture obtained by the mixing device and to convey the mixture to a filtering device;

[0273] - the filtration device being configured to retain the component associated with the polymorphonuclear cells (PBMCs) and to allow the remainder of the mixture to be eluted;

[0274] - a separation device for separating the polymorphonuclear cells (PBMC) from components associated therewith, the device being configured to facilitate the separation of the polymorphonuclear cells (PBMC) from the components;

[0275] - a processing device for the polymorphonuclear cells (PBMCs) separated by said separation device, configured in a manner allowing the Plin2 and / or Pnpla3 and / or Rab14 proteins, when present, to bind to a reagent specific for said proteins, wherein said reagent fluoresces at one or more predetermined wavelengths;

[0276] - at least one radiation source configured to emit radiation of a predetermined wavelength, for example in the form of light waves, in such a way that the agent fluoresces;

[0277] - means for acquiring an image of the irradiated sample;

[0278] - a control unit (7) connected to all said devices and said radiation source (12), programmed to control and synchronize their actuation according to an automatic mode according to predetermined operating parameters, wherein said control unit is further configured to process said images to quantify the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins present in said sample.

[0279] The control unit is further configured to compare the measured concentration with one or more reference data of Plin2 and / or Pnpla3 and / or Rab14 protein concentrations obtained from measurements on blood samples of healthy subjects and / or blood samples of the same individual collected at different times.

[0280] In one embodiment, with respect to Plin2 protein, for example, the reference data of Plin2 protein concentration is data related to healthy subjects, and wherein the control unit 7 is further programmed to detect non-alcoholic hepatic steatosis (NAFLD) condition if the data of Plin2 protein concentration obtained from the sample analysis is greater than the reference data of Plin2 protein concentration.

[0281] Similarly to what has been described above, the concentrations of Pnpla3 and Rab14 proteins can be used to detect liver fibrosis conditions and optionally assess their severity. According to a further embodiment, the reference data for the concentration of Plin2 protein are data related to the same patient and detected at a time (t0) prior to the time (tn) at which the blood sample was detected, and wherein the control unit 7 is further programmed to detect the progression of the disease over time.

[0282] For example, if the data of Plin2 protein concentration obtained from sample analysis is greater than the reference data, improvement of NAFLD is detected; if the data of Plin2 protein concentration obtained from sample analysis is less than the reference data, worsening of NAFLD is detected, and if the data of Plin2 protein concentration obtained from sample analysis is equal to the reference data, disease stagnation is detected.

[0283] Preferably, the data of Plin2 protein concentration obtained from the sample analysis is considered equal to the reference data when the difference between the two data is about 0 to about 5%.

[0284] The first and second preferred embodiments of the apparatus are Figure 4 and 5 The examples in are used to illustrate.

[0285] According to one embodiment, a blood sample, preferably already mixed with an anticoagulant, is introduced into a collection chamber 3 within the device. Preferably, the device 1 includes a puncture device 2 adapted to facilitate puncturing the skin of the individual from whom the blood sample is to be collected. For example, the puncture device 2 may be adapted to puncture the skin of a patient at a fingertip. The puncture device 2 specifically includes a protruding element, such as a needle, terminating in at least a pointed end. The puncture device 2 is carried on an outer surface of the device 1 and may be configured and articulated so as to assume a minimally obstructive configuration in a non-use state, in which the pointed element faces the outer surface of the device 1, and an in-use configuration, in which the pointed element faces away from the outer surface of the device 1. In a preferred embodiment, the puncture device 2 may include a plurality of needles having dimensions on the order of micrometers, also referred to as "microneedles." Advantageously, the microneedles may be positioned relative to one another to form a matrix of pointed elements suitable for collecting a blood sample from a patient. This arrangement of microneedles allows for multiple skin punctures to facilitate the extraction of a blood sample.

[0286] When the device according to the present description directly includes a puncture device 2 for obtaining a blood sample, the blood sample, once obtained, is collected at the aforementioned device 2 and then transferred to a collection chamber 3 inside the device 1 via a suitable fluid connection device or a transfer device 4. This transfer can be performed by aspiration of the blood sample. In particular, the aspiration is performed by the transfer device 4, which has a corresponding suction nozzle at the puncture device 2. In particular, the transfer device 4 can be a silicone diaphragm pump or a vacuum micropump. Preferably, the diaphragm pump can have the following dimensions:

[0287] - height equal to 0.6 mm;

[0288] - width equal to 5 mm;

[0289] - Length equal to 5 mm.

[0290] Therefore, the diaphragm pump is small enough to be easily moved, and the device is hardly bulky, lightweight, and easy to carry.

[0291] The means for transporting 4 may also comprise a collecting tube or cannula, such as a rubber catheter, wherein the sample may be contacted with the anticoagulant.

[0292] Alternatively, samples extracted independently by the devices described herein may be directed to the collection chamber, preferably after treatment with an anticoagulant.

[0293] In an alternative embodiment, the sample is introduced or delivered into the collection chamber 3 prior to contact with the anticoagulant.

[0294] Preferably, the device 1 is equipped with separation means 14, 5, 6, 19 of polymorphonuclear cells (PBMC) from a blood sample.

[0295] According to the present invention, the separation device 14, 5, 6, 19 may include a mixing device 14, which is preferably inserted into the collection chamber 3 and is configured to mix the blood sample with a component or component group that is easy to separate PBMCs (also defined herein as "PBMC separation device"). For example, the component group may contain a component that is easy to bind to polymorphonuclear cells (PBMCs) present in the sample and, if necessary (i.e., when the blood sample has not been treated with an anticoagulant), bind to an anticoagulant substance.

[0296] In the implementation of the device described herein, the components may be represented, for example, by suitable microbeads to which are bound antibodies against CD3+ T, PBMC-specific antigens which thus bind to PBMCs (e.g. or type technology).

[0297] The device 1 may further comprise a first s1 reservoir and / or a second s2 reservoir containing an anticoagulant and a component capable of binding to polymorphonuclear cells, respectively, each reservoir s1 , s2 preferably being provided with a respective delivery device 15 .

[0298] Furthermore, the separation device 14, 5, 6, 19 may include a moving device 5 and a filtering device 6 as described below.

[0299] As a result of the mixing, the PBMCs are combined with the aforementioned components. The mixture obtained is subjected to mechanical vibration and / or rotational stress, which is carried out by the action of a dedicated movement device 5, which is preferably contained in the collection chamber 3. In particular, the movement device 5 can be realized by at least one rotating and / or vibrating conveyor belt, on which the mixture thus obtained is conveyed.

[0300] The device 5 stresses or stirs the mixture for a predetermined duration, which can be between 5 and 20 minutes, between 5 and 15 minutes, for example a time equal to about 10 minutes. The duration can be measured, for example, by a timer T preferably connected to the same mobile device 5. Subsequently, the mixture is preferably conveyed to the filtering device 6 by the same mobile device 5, more preferably when the mobile device 5 is implemented by a conveyor belt.

[0301] According to a preferred variant embodiment, the filtering device 6 can be integrated into the moving device 5, for example in the form of a filtering conveyor belt. In other words, the operating surface of the conveyor belt (on which the mixture to be transported is placed) can be made of a filter material. The filtering device 6 is configured to retain the components bound to the PBMC and to elute the rest of the mixture, in fact resulting in the separation of the PBMC bound to the above components. For this purpose, the filtering device 6 (belonging to the group of components that are easy to separate the PBMC) has a transport port of a predetermined cross-section, for example to prevent the transport of components bound to the PBMC, but to allow the transport of the rest of the mixture.

[0302] The selection of the cutoff value of the filtration device can be easily made by a person skilled in the art based on the size of the components bound to the PBMC. In any case, the filter cutoff value should be selected in such a way that the various blood sample cells and components that are not bound to the above components are eluted and the component-PBMC complexes are retained. In one embodiment, when using microbeads conjugated to PBMC-specific anti-antigen antibodies, the filtration device will have a cutoff value selected based on the diameter of the beads. For example, for beads with a diameter of about 30 μm, the filtration device will have a cutoff value to retain the beads bound to the cells and elute all those with a lower diameter. It is desirable to use reagents already available on the market, and the filtration device 6 can be obtained by using existing reagents available on the market. technology to achieve this.

[0303] Usefully, the separation means 14, 5, 6, 19 may comprise a separation means 19 for polymorphonuclear cells (PBMCs) from components to which they are associated, which is configured to facilitate the separation of said polymorphonuclear cells (PBMCs) from said components.

[0304] Advantageously, the separation means 19 can be of the filter membrane or buffer type.

[0305] The component-PBMC complex retained on the filter means 6 is treated with a device for isolating the unique PBMCs or a separation device 19. The separation or separation of the PBMCs from the components to which they are bound can be performed, for example, using a specific buffer (separation buffer) that tends to favor the disruption of the bonds between the PBMCs and the components to which they are bound, and the apparatus can therefore include a delivery device for a suitable solution for separating the PBMCs from the components to which they are bound.

[0306] The PBMCs thus separated are transported by a collection device 9, which comprises, for example, a tube or catheter, to an analysis chamber 11. Said analysis chamber 11 preferably comprises means for exposing the PBMC cytoplasm, for example, a lysis buffer for the cell membranes. According to a variant embodiment, the cytoplasm exposure is performed directly in the collection chamber 3.

[0307] As an alternative to the solution described herein, in order to obtain PBMC separation in a blood sample, the set of components that facilitate the separation of PBMCs may include a microelectromechanical system that allows for a repulsive effect on PBMCs and thus their separation. Or microfiltration parylene membrane. The membrane can have a diameter of up to 36 mm 2 The filtration surface is about 7% to 15% with pores of different geometries (e.g., circular, oval, and rectangular) with a critical size (size) reduced to a few micrometers. Similarly, PBMCs can be isolated from blood samples by nickel electroplating techniques or by filtration through sheets of paper or plastic material.

[0308] After the PBMCs are isolated, these are processed to measure the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins using a device that makes the presence of the proteins visible and quantifiable at one or more predetermined wavelengths, such as a dye-conjugated, in particular fluorescent dye-conjugated, detection system. This can be achieved by a processing device 16, which is preferably contained in the analysis chamber 11 and is configured to perform binding of the Plin2 and / or Pnpla3 and / or Rab14 proteins to a component or signal component that exhibits the characteristics. In one embodiment, the PBMC sample is treated with a buffer for lysis of the cytoplasmic Plin2, preferably with a reagent that fluoresces at a predetermined wavelength (e.g., a fluorescent secondary antibody, such as Alexa Fluor). 488) of anti-Plin2 monoclonal antibody reaction. Therefore, the fluorescent secondary antibody is associated with Plin2. The device 1 may further include a third reservoir s3 for accommodating the antibody, which is equipped with a corresponding delivery device 15 for the antibody. In addition, the device 1 includes a spectrometer placed in the analysis chamber 11. Preferably, the spectrometer is equipped with a radiation source 12 and a device 13 for acquiring an image, both of which are, for example, placed in the analysis chamber 11 (or collection chamber 3) and facing the deposition surface for the separated and processed PBMC. The source 12 emits radiation on the deposition surface, and the radiation has a predetermined wavelength so that the fluorescent dye-conjugated antibody is visible (or presents a specific color), so that the Plin2 mode can be picked out by visual inspection. In one embodiment, the radiation source 12 is configured to emit radiation with a wavelength of 340nm to 780nm. Preferably, the radiation source can emit radiation with a wavelength of 488nm. The source 12 can include a laser microfluorimeter or other similar devices available on the market.

[0309] Advantageously, the spectrometer 11 may have the following dimensions:

[0310] - a height of 15 to 25 mm, preferably equal to 20.1 mm;

[0311] - a width of 7 to 18 mm, preferably equal to 12.5 mm;

[0312] - A depth of 5 mm to 15 mm, preferably equal to 10.1 mm.

[0313] Thus, the burden of the spectrometer 11 is reasonable and the device 1 will be easily portable.

[0314] A person skilled in the art will know the wavelength required to detect the fluorescent dye of interest based on simple information from the literature. In any case, the device can be made to emit multiple wavelengths and therefore detect multiple dyes, so the device is not therefore bound to a particular specific fluorescent dye.

[0315] Once the radiation source 12 is activated, the means for acquiring an image 13 is operated to acquire an image of the irradiated sample, wherein the Plin2 and / or Pnpla3 and / or Rab14 proteins are detectable and, most importantly, quantifiable.

[0316] The device 1 preferably includes a control unit 7, which is connected to all of the above-mentioned devices and components and is programmed to control and synchronize their activation according to an automatic mode according to predetermined operating parameters. The operating parameters may include the activation duration of each of the above-mentioned devices / components, as well as the blood sample pressure and / or aspiration rate, its delivery rate, the amount of anticoagulant / PBMC separation component / fluorescent reagent / anti-Plin2 antibody delivered from the corresponding reservoir s1, s2, s3, and / or the wavelength of the radiation source. Similarly, the control unit 7 may include or be connected to an interface device 8, which is configured to allow a user to control the activation of the device 1 and / or modify / select the above-mentioned operating parameters. The interface device 8 may include at least one power button for the device 1.

[0317] In addition, the control unit 7 may include an image processing microprocessor that is programmed to identify the presence of Plin2 and / or Pnpla3 and / or Rab14 and quantify the protein in the image acquired by the device 13. Similarly, the microprocessor may be programmed to quantify the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins detected in the acquired image and output said data. The output data may be transmitted to an external electronic device, for example, via a wired or wireless connection device included in the device 1 itself, or to a display device that may be integrated in the above-mentioned interface device 8, for example, including a display.

[0318] The device 1 of the present invention, in particular the microprocessor of the control unit 7, can further preferably be programmed to automatically compare the data of the Plin2 protein concentration obtained by analyzing the images acquired by the device 13 with reference data of the Plin2 and / or Pnpla3 and / or Rab14 protein concentration, which reference data can be Plin2 and / or Pnpla3 and / or Rab14 related to healthy subjects (hereinafter referred to as reference data for simplicity). In this case, the device has a dedicated diagnostic function.

[0319] The above-mentioned reference data can also be stored in a storage module, for example contained in the central unit 7 and connected to the microprocessor, in which data on the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins previously detected by the device itself for the same patient can also be stored and analyzed in order to track the evolution of the disease or evaluate the effectiveness of therapeutic treatment.

[0320] In particular, the interface device may be configured to display a graph in which the pattern of detected Plin2 and / or Pnpla3 and / or Rabl4 concentration values ​​as a function of time is shown.

[0321] Furthermore, the interface device 8 may be configured to automatically display a visual signal according to a predetermined color code and / or reproduce an acoustic signal based on a comparison result of the data of Plin2 protein and / or Pnpla3 and / or Rab14 concentration obtained by analysis with reference data.

[0322] For example, with reference to Plin2 protein; when monitoring disease progression, if the data of the Plin2 protein concentration obtained from the sample analysis is less than the reference data, improvement of NAFLD is detected, which can correspond to turning on the green light. Conversely, if the data of the Plin2 protein concentration obtained from the sample analysis is greater than the reference data, worsening of NAFLD is detected, which can correspond to turning on the red light and / or the reappearance of the acoustic alarm. Similarly, if the data of the Plin2 protein concentration obtained from the sample analysis is equal to the reference data, disease stagnation is detected, which can correspond to turning on the yellow light.

[0323] In the case of diagnostic applications, a similar system may be used.

[0324] Furthermore, different reference data can be modified and / or selected via the same interface device 8, and / or the light / sound signal display / automatic reproduction settings can be modified.

[0325] Preferably, the device 1 is implemented to be portable, so as to allow the measurement of Plin2 and / or Pnpla3 and / or Rab14 protein concentrations also in locations distributed relative to a hospital or medical center. In particular, the device 1 may include its own power supply means allowing it to be independent, such as a rechargeable battery and / or a photovoltaic or wind energy recharging device.

[0326] The designed device has many advantages related to the dimensions of the various components, in particular the dimensions of the puncture device 2, the spectrometer and the pump 4. The combination of said components in such dimensions allows the portable device 1 to be easy to carry and use, being lightweight and reducing burden.

[0327] It will be understood how the portable device described can be adapted to allow, in addition to the measurement of Plin2 protein concentration, also the measurement of concentrations of other protein types in serum / plasma or PBMCs, with appropriate adaptation and necessary modifications being within the capabilities of a person skilled in the art.

[0328] In this regard, the device 1 may comprise a connection unit, preferably a "plug-in" unit, configured to adapt the device to other biomarkers.

[0329] According to an alternative embodiment, the device may comprise a plurality of analysis chambers 11 separated between them, each chamber being configured to measure the concentration of a different protein in a blood sample, said proteins being selected from the group consisting of: Plin2, Pnpla3 and Rabl4.

[0330] The device is therefore versatile, capable of determining the concentration of different proteins and thus allowing the determination of the occurrence and grade of different pathologies as described above.

[0331] Preferably, the apparatus may include a selection device operatively connected to the control unit 7 and configured to route the blood sample to the correct analysis chamber 11 selected based on the protein concentration to be determined. The selection device may be controlled by the control unit 7, which is configured to transmit input data containing information about the protein concentration to be determined. Based on the input data, the selection device transfers the blood sample to a specific analysis chamber 11.

[0332] Advantageously, the device may comprise at least one processing unit configured to implement the computer program according to one or more of the examples reported below.

[0333] Usefully, the processing unit may be operatively connected to the control unit 7. Thus, the device may use the data detected in the analysis chamber 11 to implement the computer program described below.

[0334] The apparatus of the present invention may be implemented with a suitable computer program, for example, as defined below depending on the intended use and the method one wishes to perform among those described and claimed herein.

[0335] The object of the present invention is therefore the computer program listed below:

[0336] A computer program for diagnosing NAFLD or NASH in an individual to be analyzed, comprising a list of instructions. When the list of instructions is executed on an electronic computer, provided with a first concentration value of Plin2 protein in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, and a second value C0 and a third value C1 of the protein concentration as defined in the present specification, the program performs the following steps:

[0337] comparing the first value with the second and third values, and

[0338] NAFLD is diagnosed when the first value is greater than or equal to the value C0 and less than the value C1, or NASH is diagnosed when the first value in the blood sample is greater than or equal to the value C1.

[0339] A computer program for diagnosing the NAS score in an individual to be analyzed, comprising an instruction list. When the instruction list is executed on an electronic computer, provided with a first concentration value of Plin2 protein in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, and a second value C3, a third value C4, and a fourth value C5 of the Plin2 protein concentration as defined herein, the computer program performs the following steps:

[0340] The first value is compared with the second value, the third value, and the fourth value, and the NAS score is diagnosed as:

[0341] When the first value is greater than or equal to the cutoff value C2 and less than or equal to the cutoff value C3, the NAS score is 1.

[0342] When the first value is greater than the value C2 and less than or equal to the cutoff value C4, the NAS score is 2,

[0343] When the first value is greater than the value C4, the NAS score is 3.

[0344] A computer program for diagnosing the severity of NASH in an individual to be analyzed comprises an instruction list. When the instruction list is executed on an electronic computer, provided with a first concentration value of Plin2 protein in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, and a second value C5, a third value C6, and a fourth value C7 of Plin2 protein concentration as defined herein, the computer program performs the following steps:

[0345] The first value is compared with the second value, the third value, and the fourth value and the severity level of NASH is diagnosed as:

[0346] When the first value is greater than the value C5 and less than the value C6, mild,

[0347] When the first value is greater than the value C6 and less than the value C7, moderate, and

[0348] When the first value is greater than the value C7, it is serious.

[0349] A computer program for diagnosing liver fibrosis in an individual to be analyzed, comprising an instruction list. When the instruction list is executed on an electronic computer, provided with a first concentration value of Pnpla3 and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample and a second concentration value C8 of Pnpla3 and Rab14 proteins as defined in the above embodiment, the computer program performs the following steps:

[0350] comparing the first value to the second value, and

[0351] When the first value is greater than C8, liver fibrosis is diagnosed.

[0352] A computer program for diagnosing the stage of liver fibrosis in an individual to be analyzed, comprising a list of instructions. When the list of instructions is executed on an electronic computer, provided with a first concentration value of Pnpla3 and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, and a second value C9, a third value C10, and a fourth value C11 of the Pnpla3 and Rab14 protein concentrations as defined in the present specification, the computer program executes the following steps:

[0353] The first value is compared with the second value, the third value, and the fourth value and the fibrosis score stage is diagnosed as:

[0354] When the first value is greater than or equal to the value C9 and less than the value C10, stage 1,

[0355] When the first value is greater than or equal to the value C10 and less than or equal to the value C11, stage 2, and

[0356] When the first value is greater than the value C11, stage 3.

[0357] Alternatively, the computer program for diagnosing NAFLD, NAS score, NASH, or severity level of NASH may include one of the two instruction lists for diagnosing liver fibrosis and / or liver fibrosis stage.

[0358] The present invention also relates to a computer program for monitoring the effectiveness of a therapeutic treatment of NAFLD or NASH in a patient, comprising a list of instructions which, when executed on an electronic computer, is provided with a first concentration value of the Plin2 protein in a blood sample of the patient or in a sample of leukocytes extracted from the blood sample at a time t0 and a second concentration value of the Plin2 protein in a blood sample of the patient or in a sample of leukocytes extracted from the blood sample at a time after t0, performs the following steps:

[0359] comparing the first value to the second value, and

[0360] If the second value is less than the first value, effectiveness of the therapeutic treatment is detected;

[0361] And a computer program for monitoring the progression of NAFLD or NASH in a patient, comprising an instruction list, which, when executed on an electronic computer, is provided with a first concentration value of Plin2 protein in a blood sample of the patient or in a white blood cell sample extracted from the blood sample at time t0 and a second concentration value of Plin2 protein in the blood sample of the patient or in a white blood cell sample extracted from the blood sample at a time after t0, performs the following steps:

[0362] comparing the first value to the second value, and

[0363] If the second value is less than the first value, an improvement in NAFLD or NASH is detected,

[0364] If the second value is greater than the first value, worsening of NAFLD or NASH is detected,

[0365] If the second value is approximately equal to the first value, stagnation of NAFLD or NASH is detected.

[0366] The invention is directed to a device in any of the embodiments described above and as claimed, further comprising at least one processing unit configured to implement a computer program according to one or more of the examples described above.

[0367] Usefully, the processing unit may be operatively connected to the control unit 7. Thus, the device may use the data detected in the analysis chamber 11 to implement the above-mentioned computer program.

[0368] The present invention also aims to use the device in any of the above embodiments to implement the method of the present invention.

[0369] Finally, the object of the present invention is a method of treatment for treating patients suffering from NAFLD, said patients being subjected to the monitoring described herein.

[0370] Example

[0371] method

[0372] Nineteen obese subjects of both sexes with NAFLD (10 females and 9 males, mean age 38.5 ± 1.9 years, BMI 36.79 ± 4.43 kg / m 2 ) were enrolled and underwent bariatric surgery. Peripheral venous blood was collected and supplemented with EDTA after a 12-hour overnight fast at enrollment. The patients provided written informed consent for the study and surgical intervention.

[0373] Medical records of all subjects were collected. Objective examinations including height, weight, and anthropometric measurements were also performed. Body mass index (BMI) was calculated as weight (kg) divided by height (m 2 ) were calculated. A detailed list of medications used was collected.

[0374] Exclusion criteria were: (1) frequent and / or excessive alcohol consumption (>20 g alcohol / day for women and >30 g alcohol / day for men); (2) clinical evidence of NAFLD secondary to iatrogenic gastrointestinal or immunodeficiency (HIV infection); (3) clinical evidence of non-NAFLD liver disease, including hepatitis B or C, or hemochromatosis, (4) Wilson's disease, (5) glycogenolysis, (6) α-1 antitrypsin deficiency, (7) autoimmune hepatitis, (8) cholestatic liver disease, (9) presence of associated cardiovascular, gastrointestinal, or respiratory disease, or any hormonal disturbance, (10) clinical evidence of decompensated liver disease (Child-Pugh score >7), (11) current narcotic abuse, (12) associated systemic disease, and (13) pregnancy.

[0375] A liver biopsy was obtained during surgery.

[0376] All subjects underwent an oral glucose tolerance test (OGTT) and collected data at 0, 30, 60, 90, 120, 150 and 180 minutes to measure blood insulin and sugar levels (blood glucose). Insulin sensitivity was measured as OGIS (the acronym for Oral Glucose Insulin Sensitivity), a method for calculating insulin sensitivity from an OGTT. The index generated by OGIS is similar to the insulin sensitivity index (Mari A, Pacini G, Brazzale AR, Ahrén R. Comparative evaluation of simple insulin sensitivity methods based on the oral glucose tolerance test. Diabetologia 2005; 48: 748-751) obtained using a clamp.

[0377] Liver histology

[0378] Liver biopsy fragments placed under formalin were thoroughly washed with 60% isopropyl alcohol, which was then evaporated. Oil Red O (ORO) solution was added for 10 minutes, then removed and the samples washed four times with water. After removing the Oil Red O, the fragments were incubated in 100% isopropyl alcohol. Mononuclear cells were also stained with ORO in the same manner.

[0379] The samples were then observed under an LSM 510 confocal microscope equipped with appropriate filters.

[0380] In addition, a portion of the biopsy material obtained during surgery was mounted on storage slides, prepared from fragments fixed in 10% formalin, embedded in paraffin blocks, and stained with hematoxylin and eosin, to assess the percentage of steatosis. Slide readings were performed by an expert liver anatomic pathologist under blinded conditions.

[0381] The Brunt classification (Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999; 94: 2467–2474) is used to assess histological scores and NAFLD / NASH (nonalcoholic steatohepatitis) stages. Specifically, steatosis is defined as the following scores based on fat in the lobule: 0, none (<1%); 1, 1%-25%; 2, 26%-50%; 3, 51%-75%; and 4>75%. Inflammation is scored as follows: 1, mild (lymphocytes are isolated (scattered) or aggregated into small structures within the portal area and lobule); 2, moderate (as grade 1, but with larger portal area and lobular infiltration); and 3, severe (same as grade 2, but with more severe inflammation). The fibrosis stage was as follows: 0, absent; 1, pericellular in the centrilobular area; 2, periportal and pericellular; 3, bridging fibrosis; and 4, cirrhosis.

[0382] Monocyte isolation

[0383] PBMCs were obtained from whole blood by standard gradient centrifugation on Ficoll-Hypaque (GE Healthcare Bio-Sciences, Piscataway, NJ). PBMCs were then washed and monocytes were isolated using the Pan Monocyte Isolation Kit.

[0384] Hepatocyte isolation

[0385] The liver biopsy fragments were minced and washed in HBSS to remove blood. The tissue was then transferred to a 50 ml test tube containing EGTA buffer (HBSS, 0.5 mM EGTA, 0.5% BSA) and stirred at 100 rpm for 10 minutes in a water bath maintained at 37°C. The tissue was then placed in digestion buffer (HBSS, 0.05% collagenase IV, 0.5% fatty acid-free BSA, 10 mM CaCl2) and stirred at 100 rpm for 10 minutes in a water bath maintained at 37°C. The supernatant was collected and filtered through a 100 μm pore filter, and the cell suspension was centrifuged at 80 rpm for 5 minutes at 4°C, and the supernatant was discarded.

[0386] Lipid droplet staining

[0387] The isolated monocytes and hepatocytes were incubated with 4% formalin for 20 minutes and stained with Nile red (100 ng / mL). The cell nuclei were stained with DAPI.

[0388] Pictures were taken with a Spinning Disk confocal microscope; Crest X-Light Confocal Imager (Germany), and images were analyzed with MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices).

[0389] Plin2 measurement

[0390] Mononuclear cells and liver biopsies were homogenized in RIPA buffer containing a protease inhibitor cocktail. The homogenate was centrifuged at 13,000 rpm for 30 minutes at 4°C. Protein content was measured using the Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA). Protein lysates (30 μg) were separated on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight with anti-Plin2 antibodies (LS-BIO, Seattle, WA) and anti-β-actin. Qualitative and quantitative analyses were performed using the Chemidoc XRS Image system and Image Lab 5.0 software (Bio-Rad Laboratories, Hercules, CA). All data were normalized to β-actin levels (8H10D10).

[0391] statistics

[0392] When not otherwise stated, data are expressed as mean ± SD. Spearman correlation is used to measure the degree of correlation between two variables. The consistency between the two methods is measured by Bland-Altman diagram. Bland-Altman results show the difference and mean values ​​obtained using the two methods (Plin2 levels in monocytes and liver). The standard deviation (SD) of the difference between the two methods is SD deviation. The consistency limits are measured as mean deviation, i.e., SD deviation of the mean ± 1.96 of the difference between the two measurement methods.

[0393] The sensitivity and specificity of the method were investigated using neural network analysis. The neural network analysis parameters are reported below: Two input layers were used: Plin2 in monocytes and subject age. There was only one hidden layer with six units and a hyperbolic (tangent) activation function. The output layer was the dependent variable, namely the NAS level. The activation function was Softmax, and the error function was cross-entropy.

[0394] Neural network analysis provides the area under the curve (AUC) of the ROC (receiver operating characteristic) curve. The ROC curve has sensitivity on the x-axis and the number of false positives (1 minus specificity) on the y-axis. The test has the best predictive value when the AUC is closest to 1. Statistical analysis was performed using SPSS version 13.

[0395] result

[0396] The grade of hepatic steatosis was assessed by liver biopsy and the NAFLD activity score (NAS) as defined above. The histological method defines the NAFLD grade. The mean steatosis grade of all examined patients was 2.42±1.17, and the subjects showed steatosis grades of 1 to 4.

[0397] To demonstrate that ectopic fat accumulation also occurs in monocytes, liver biopsies and monocytes were stained with Nile red ( Figure 1 , Panel A). In monocytes, lipids aggregate into larger droplets.

[0398] Plin2 expression in monocytes was strongly positively correlated with Plin2 expression in the liver (R=0.84P<0.0001). An example of Plin2 protein expression by Western blotting is reported in Figure 1 In the small picture B.

[0399] on the contrary, Figure 2 reported ORO staining in liver sections and mononuclear cells from the same subjects.

[0400] The Plin2 level in the liver was well correlated with the grade of hepatic steatosis (R=0.91, P<0.001), and the Plin2 level in monocytes was well correlated with the grade of hepatic steatosis (R=0.89, P<0.001).

[0401] Figure 3 Reported the Bland-Altman diagram comparing two methods (i.e. Plin2 levels in monocytes and liver). The difference between the values ​​obtained by the two measurements is reported on the Y-axis, and the mean value is on the Y-axis. The dotted line has reported 95% confidence limits between the two measurements. This figure shows that all points are within the confidence limits, meaning that the method of the present invention is effective.

[0402] It was also found that the median fluorescence intensity (MFI, Figure 4 There was a good correlation between the Plin2 levels expressed by ) and those measured by Western blotting (R=0.92; P<0.0001).

[0403] The Plin2 level (MFI) in PBMCs was 48.88±5.80 (SEM), showing an excellent correlation with the level in monocytes 42.90±5.77 (SEM) (R=0.98, P=0.0004).

[0404] As evidence of a correlation between the severity of nonalcoholic hepatic steatosis and Plin2 levels, it was found that Plin2 levels in the liver and monocytes were negatively correlated with insulin sensitivity expressed as OGIS, and the regression equation for monocytes was:

[0405] OGIS=-44.59*Plin2+411.3

[0406] where R 2 was 0.91, and P < 0.0001

[0407] OGIS is the acronym for Oral Glucose Insulin Sensitivity Index, measured in ml x min- 1 x m- 2 Plin2 was measured by Western blotting in units relative to the β-actin reference protein.

[0408] Analysis of patients

[0409] Ninety-one participants underwent liver biopsy for suspected NASH, while 21 participants (control group) undergoing elective cholecystectomy with a normal body mass index and negative liver ultrasound underwent liver core biopsy during surgery. Participants ranged in age from 18 to 67 years, and 55% were female and 45% were male.

[0410] Histological examination highlighted various stages of NAFLD activity score (NAS).

[0411] Plin2 levels in circulating monocytes analyzed by cytofluorimetry were used to predict NAS.

[0412] In the controls, some had NAS=0 and others had NAS=1.

[0413] Cytofluorimetry

[0414] Monoclonal antibody against Plin2 was obtained from LS-BIO (Seattle, WA), fluorophore-conjugated secondary monoclonal antibody Alexa Fluor 488 was from Life Technology (Carlsbad, CA), and anti-CD14-ECD antibody was from Beckman Coulter (Brea, CA).

[0415] Monocytes were identified by using anti-CD14-ECD antibody for identification of the mononuclear cell population among all polymorphonuclear cells.Mononuclear cells were fixed and permeabilized by standard techniques as reported below.

[0416] Mix 1 part Fixation / Permeabilization Concentrate with 3 parts Fixation / Permeabilization Diluent. Resuspend the cell pellet (2 x 106 cells) in 300 μL of 1X Permeabilization Buffer and incubate at +4°C for 45 minutes. Wash with 1X phosphate-buffered saline (PBS), centrifuge, and resuspend the cells in 100 μL of 1X PBS. Divide the suspended cells into two tubes (50 μL each).

[0417] Tube 1: Suspension cells were stained with IgG antibody Alexa Fluor 488 (1:2000) for 20 minutes in room temperature and darkness. This tube serves as a negative control.

[0418] Tube 2: Suspension cells were stained with anti-Plin2 (1 μl in 50 μl), followed by staining with IgG antibody Alexa Fluor 488 and anti-CD14-ECD antibody (4 μl in 50 μl) for 20 minutes in room temperature and darkness.

[0419] They were washed, centrifuged, and resuspended in 500 μL of 1X PBS before cytofluorimetric analysis.

[0420] Therefore, monocytes were stained for Plin2 using an anti-Plin2 monoclonal antibody and a fluorophore-conjugated secondary antibody (Alexa Fluor 488) that binds to the anti-Plin2 antibody.

[0421] The instrument used for cytofluorimetry was an FC 500 (Beckman Coulter, Brea, CA), and the data were analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0422] Plin2 detection is achieved via a fluorescent tracer that generates a signal that is picked up by a cytofluorimeter photodiode and converted to mean fluorescence intensity (MFI).

[0423] The MFI is proportional to the amount of antibody that recognizes and binds to the cellular antigen (in this case, Plin2), thereby enabling protein quantification (Mizrahi O., Shalom EI, Baniyash M., Klieger Y. Quantitative flow cytometry: concerns and recommendations in clinic and research. Cytometry BClin Cytom. 2017).

[0424] Histologically, NAFLD is defined as steatosis affecting more than 5% of hepatocytes, while NASH is defined as the presence of any grade of hepatocellular ballooning and lobular inflammatory infiltrates in addition to steatosis, regardless of their number. The NAFLD Activity Score (NAS) is generated by combining the steatosis, hepatocellular ballooning, and lobular inflammatory infiltrates in liver tissue. As can be seen in the table below, the highest score generated by summing the individual components is 8.

[0425]

[0426]

[0427] (Kleiner DE, Brunt EM, Van Natta M., Behlinh C., Contos MJ, Cummings O. W., Ferrell LD, Liu Y.-C., Torbenson MS, Unalp-Arida A., Yeh M., McCullough A. J., Sanyal AJ for the Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system fornonalcoholic fatty liver disease.Hepatology 41:1313-1321,2005)

[0428] The levels of Plin2 (mean fluorescence intensity [MFI]) at different NAS stages are reported in the following table:

[0429]

[0430] As described below, the correlation between mean Plin2 levels (MFI) and NAS stage was very good.

[0431] The independent variable on the X-axis is the mean of Plin2, while the dependent variable is the NAS stage ( Figure 7 ).

[0432]

[0433] The independent variable is mean_Plin2

[0434] R 2 The value was very high (0,985), with a significance of P < 0.0001, indicating that the model for predicting the histological stage of NASH (ie, NAS) was very good.

[0435]

[0436] The independent variable is mean_Plin2

[0437] The ability of Plin2 to predict various NAS stages was investigated through neural network analysis.

[0438] The neural network analysis parameters are reported below. Two input layers were used: Plin2 in monocytes and subject age. There was only one hidden layer with six units and a hyperbolic (tangent) activation function. The output layer was the dependent variable, the NAS layer. The activation function was Softmax, and the error function was cross-entropy.

[0439] Network Information

[0440]

[0441] a. Exclude deviation units

[0442] The design predicted incorrect NAS values ​​in only 6.3% of cases during training and in 11.35% of cases during 0.05-second testing, as reported below.

[0443] Model Overview

[0444]

[0445] Dependent variable: NAS

[0446] a. Error calculation based on test samples

[0447] The data showed that the area under the receiver operating characteristic (ROC) curve (AUC) was very high, ranging from a minimum of 0.974 to a maximum of 1.

[0448] All stages were predictable with high sensitivity and specificity.

[0449] Area under the curve of ROC analysis

[0450]

[0451] The importance of the Plin2 variable in the model is reported below and was 68.3%, but reached 100% when normalized, while the importance of the second variable used in the model (ie, age) was 31.7% and 46.3% when normalized.

[0452] Importance of independent variables

[0453]

[0454] (See Figure 8 )

[0455] Multiple comparisons analysis adjusted for repeated inferences (i.e., multiple comparisons) demonstrated high significance of the differences between the different NAS stages, as shown in the table below.

[0456] ANOVA

[0457] Monocyte Plin2

[0458]

[0459] Multiple comparisons

[0460] Dependent variable: Monocyte Plin2

[0461]

[0462]

[0463]

[0464] *.The mean difference is significant at the 0.05 level

[0465] In conclusion, Plin2 in monocytes is highly predictive of NASH severity stage (i.e., NAS score) and enables peripheral blood testing to replace invasive tests such as biopsy.

[0466] fibrosis

[0467] The levels of Patatin-like phospholipase domain-containing protein 3 (Pnpla3) (Sigma Aldrich SAB1401851) and Ras-related protein Rab-14 (Sigma Aldrich R0656), which are normally found in monocytes, were determined by cytofluorimetry and histological examination of liver and monocyte isolation in a total of 132 subjects.

[0468] The mean values ​​(MFI) for each stage of SAF fibrosis from F0 to F4 were as follows (Bedossa P, Poitou C, Veyrie N, Bouillot JL, Basdevant A, Paradis V, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology 2012; 56: 1751–1759).

[0469]

[0470] ANOVA

[0471]

[0472] A neural network analysis was then performed to calculate the AUC of the ROC curve using monocyte Pnpla3 and monocyte Rab14 as independent variables, NAS as the dependent variable, and the presence or absence of diabetes as a covariate (binary variable, yes = 1, no = 0). These variables were used in the input layer of the neural network. There was only one hidden layer with 8 units, and the activation function was hyperbole. The output of the model was the fibrosis grade (SAFF), the activation function was softmax, and the error function was cross entropy.

[0473] Case Progress Overview

[0474]

[0475] Network Information

[0476]

[0477] a. Exclude deviation units

[0478]

[0479] Dependent variable: SAF F

[0480] a. Error calculation based on test samples

[0481] During model training, the error percentage was 14.3%, and during testing it was 0% testing, taking 0.05 minutes.

[0482] The area under the curve (AUC) of the ROC analysis is reported in the following table:

[0483] ROC AUC

[0484]

[0485] Thus, stage 0 (ie, no fibrosis) was predicted at 100%, stage 1 at 95%, stage 2 at 100%, and stage 3 at 95.5%.

[0486] Importance of the independent variables (monocyte Pnpla3 and monocyte Rab14) in the model: the importance of monocyte Pnpla3 was 62%, but it was 100% when normalized; the importance of monocyte Rab14 was 29.7%, which reached 47.9% after normalization; and the importance of the presence or absence of diabetes was 0.84%, which was 13.5% after normalization.

[0487] Importance of independent variables

[0488]

[0489] (See Figure 9 )

[0490] Therefore, the use of Pnpla3 and Rab14 can provide very accurate information on fibrosis.

[0491] However, Plin2 alone also gave a good indication of fibrosis.

[0492] Case Progress Overview

[0493]

[0494] Network Information

[0495]

[0496] a. Exclude deviation units

[0497] Model Overview

[0498]

[0499] Dependent variable: SAF F

[0500] a. Error calculation based on test samples

[0501] The percentage of incorrect predictions during training is: 14.3% and during testing is: 20%.

[0502] The ROC AUCs of Plin2 for defining fibrosis reported below were 98.9% for stage 0, 94% for stage 1, 97% for stage 2, 98.8% for stage 3, and 100% for stage 4 of fibrosis.

[0503] Area under the curve

[0504]

[0505] The importance of Pin2 in monocytes in diagnosing fibrosis was 49.2% and 96.8% after normalization, and the importance of the presence or absence of diabetes was 50.8% and 100% after normalization, as shown in Tables 2 and 3. Figure 10 reported.

[0506] Importance of independent variables

[0507]

[0508] ANOVA

[0509] Monocyte Plin2

[0510]

[0511] Multiple comparisons

[0512] Dependent variable: Monocyte Plin2

[0513]

[0514]

[0515]

[0516] *.The mean difference is significant at the 0.05 level.

Claims

1. Use of the reagent in preparing a kit for diagnosing liver fibrosis, The reagent is used to measure the concentration values ​​of Plin2, Pnpla3 and Rab14 proteins in monocytes.

2. The method according to claim 1, wherein the concentration of the Plin2 protein is measured by Western blotting, cytofluorimetry, ELISA, or quantitative PCR. 3 . The method according to claim 2 , wherein the concentration of the Plin2 protein is measured by cytofluorimetry using a monoclonal antibody that specifically binds to Plin2 and does not bind to other proteins and is labeled with a suitable fluorescent dye.

4. The use according to claim 1, wherein the concentration values ​​of Pnpla3 and Rab14 proteins are measured by Western blotting, or cytofluorimetry, or ELISA, or quantitative PCR.

5. The use according to claim 4, wherein the concentration values ​​of Pnpla3 and Rab14 proteins are measured by cytofluorimetry using a monoclonal antibody that specifically binds to Pnpla3 and does not bind to other proteins and a monoclonal antibody that specifically binds to Rab14 and does not bind to other proteins, both labeled with appropriate fluorescent dyes.

6. A computer program product for diagnosing liver fibrosis in an individual to be analyzed, comprising an instruction list, wherein when the instruction list is executed on an electronic computer, provided with a first concentration value of each of Plin2, Pnpla3, and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample; and a cutoff value C8 of Pnpla3 or Rab14 protein between patients with liver fibrosis and healthy individuals, the computer program product performs the following steps: comparing said first concentration value of Pnpla3 and / or Rab14 to said C8, and When the first concentration value of Pnpla3 and / or Rab14 is greater than C8, liver fibrosis is diagnosed.

7. A computer program product for diagnosing the severity level of liver fibrosis in an individual to be analyzed, comprising an instruction list, which, when executed on an electronic computer, is provided with a first concentration value of each of Plin2, Pnpla3, and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, a cutoff value C9 for the concentration of Pnpla3 and Rab14 proteins between a healthy individual and a patient with stage 1 liver fibrosis, a cutoff value C10 for the concentration of Pnpla3 and Rab14 proteins between a patient with stage 1 liver fibrosis and a patient with stage 2 liver fibrosis, and a cutoff value C11 for the concentration of Pnpla3 and Rab14 proteins between a patient with stage 2 liver fibrosis and a patient with stage 3 liver fibrosis, and performs the following steps: The first concentration value of Pnpla3 and / or Rab14 is compared with C9, C10, and C11, and the stage of liver fibrosis is diagnosed as: When the first concentration value of the Pnpla3 and / or Rab14 is greater than or equal to the C9 and less than the C10, stage 1, When the first concentration value of the Pnpla3 and / or Rab14 is greater than or equal to the C10 and less than or equal to the C11, stage 2, and When the first concentration value of the Pnpla3 and / or Rab14 is greater than the C11, stage 3.

8. A computer program product for diagnosing liver fibrosis in an individual to be analyzed, comprising a list of instructions, which, when executed on an electronic computer, is provided with a first concentration value of each of Plin2, Pnpla3, and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, wherein the first concentration value is measured by cytofluorimetry using monoclonal antibodies labeled with a suitable fluorescent dye that specifically bind to Plin2, Pnpla3, and Rab14, respectively, and do not bind to other proteins; and a cutoff value of the Pnpla3 and / or Rab14 protein concentration equal to 1.24 MFI expressed as mean fluorescence intensity (MFI), and performs the following steps: comparing the first concentration value of Pnpla3 and / or Rab14 with a cutoff value of Pnpla3 and / or Rab14, Liver fibrosis is diagnosed when the first concentration value of Pnpla3 and / or Rab14 is greater than or equal to the cutoff value of Pnpla3 and / or Rab14 of 1.24 MFI.

9. A computer program product for diagnosing the severity of liver fibrosis in an individual to be analyzed, comprising a list of instructions, which, when executed on an electronic computer, is provided with first concentration values ​​of each of Plin2, Pnpla3, and Rab14 proteins in a blood sample of the individual to be analyzed or in a leukocyte sample extracted from the blood sample, wherein the first concentration values ​​are measured by cytofluorimetry using monoclonal antibodies labeled with appropriate fluorescent dyes that specifically bind to Plin2, Pnpla3, and Rab14, respectively, and do not bind to other proteins; and cutoff values ​​of Pnpla3 and / or Rab14 protein concentrations expressed as mean fluorescence intensity (MFI) equal to 1.24 MFI, 2.3 MFI, and 3.10 MFI, and performs the following steps: comparing the first concentration value of Pnpla3 and / or Rab14 with a cutoff value of Pnpla3 and / or Rab14, When the first concentration value of Pnpla3 and / or Rab14 is greater than or equal to the cutoff value of Pnpla3 and / or Rab14 of 1.24 MFI and less than the cutoff value of Pnpla3 and / or Rab14 of 2.4 MFI, it is diagnosed as mild stage 1 liver fibrosis, when the first concentration value of Pnpla3 and / or Rab14 is greater than or equal to the cutoff value of Pnpla3 and / or Rab14 of 2.4 MFI and less than the cutoff value of Pnpla3 and / or Rab14 of 3.10 MFI, it is diagnosed as stage 2 liver fibrosis, and when the first concentration value of Pnpla3 and / or Rab14 is greater than or equal to the cutoff value of Pnpla3 and / or Rab14 of 3.10 MFI, it is diagnosed as stage 3 liver fibrosis.

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