Novel pathological markers and their uses

Abstract

The present invention relates to novel methods for diagnosing non-alcoholic fatty liver disease (NAFLD) or monitoring its progression by identifying new pathological markers, detecting and quantifying said markers, and devices enabling said methods to be carried out.

Description

[Technical Field]

[0001] The present invention relates to novel methods for diagnosing or monitoring the progression of non-alcoholic fatty liver disease NAFLD and / or NASH by identifying new pathological markers, detecting and quantifying (qualifying) said markers, and to devices enabling said methods to be carried out. [Background technology]

[0002] Non-alcoholic fatty liver disease (NAFLD), also referred to by the acronym Non-Alcoholic Fatty Liver Disease, is a spectrum of liver tissue degeneration characterized by excessive accumulation of fat within hepatocytes (steatosis, consisting of an accumulation of more than 5% of cellular triglycerides) in the absence of heavy alcohol consumption and secondary causes of liver damage.

[0003] NAFLD currently constitutes the leading cause of degenerative hepatolytic hepatitis index in the Western world in adults and, surprisingly, in children and teenagers as well.

[0004] Updated data reported in 2016 by Bugianesi and Marietti (Recenti Prog Med 2016;107:360-368) indicate that the global prevalence of NAFLD is 25% among the adult population and that among individuals with NAFLD, the global prevalence of NASH (nonalcoholic fatty liver disease) diagnosed by liver biopsy is 20-50%. The rates reported herein are dramatically elevated in at-risk patients, e.g., those with obesity and / or type 2 diabetes.

[0005] The most striking epidemiological data concern the pediatric population, in which obesity and metabolic syndrome have been progressively increasing worldwide, and to date this has been evident in Europe, with estimates similar to those recorded in the U.S. Data recorded between 2007 and 2010 reveal that the prevalence of NAFLD is equal to approximately 7%, a value three times higher than that recorded just under 20 years earlier.

[0006] While simple fatty liver carries a negligible risk for progression to cirrhosis, a significant proportion (10-15%) of subjects with NAFLD exhibit the histological features of necroinflammation and swelling that characterize the most severe form of liver disease, nonalcoholic steatohepatitis (NASH), which can progress to associated complications including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

[0007] It is a complex and multifactorial condition, and the severity and onset of nonalcoholic fatty liver disease are regulated by both genetic and environmental factors. It is frequently associated with conditions such as metabolic syndrome and type 2 diabetes, but it also plays a role in the onset, associated development, and complications of the syndrome. Given that the increasing prevalence of NAFLD is associated with dietary and social changes in developed countries, this condition has potentially significant clinical implications. For example, in a significant number of cases, this condition can develop into nonalcoholic fatty liver disease, which is also the cause of cirrhosis and HCC. Therefore, it is clearly important to be able to identify high-risk subjects early and accurately in order to be able to investigate and predict the onset of complications that may affect the prognosis in hepatic and extrahepatic (cardiovascular) values.

[0008] Besides liver damage, the major cause of morbidity and mortality in individuals suffering from NAFLD is in fact represented by cardiovascular complications, which are more prevalent in these individuals than in the general population, regardless of the presence of type 2 diabetes and other risk factors.

[0009] As Bugianesi and Marietti further reported in 2016, since the diagnosis of NASH is currently contingent on the performance of a liver biopsy, in recent years, efforts by the scientific community have been directed towards searching for large-scale applicable and relevant genetic polymorphisms for non-invasive markers of liver damage in order to precisely address screening programs, follow-up and treatment efforts.

[0010] Because biopsies of this organ are typically performed only when a severe syndrome is present, identifying a noninvasive diagnostic method would allow timely diagnosis in subjects who would not normally undergo liver biopsy. Furthermore, noninvasive diagnostics could also monitor the effectiveness of therapeutic treatments administered to patients over time. It should be noted that ultrasound-guided liver biopsies are expensive tests that must be performed in hospitals, with costs ranging from $2,000 to over $7,000. Mortality rates from fatal hemorrhage after liver biopsy remain in the 0.13% to 0.33% range and may be even higher in subjects with altered clotting time.

[0011] Therefore, there is currently a great need to select diagnostic markers for NAFLD that allow accurate and rapid diagnosis without the need for liver biopsy intervention, thereby enabling the expansion of disease diagnosis in the population due to the non-invasive nature of the diagnostic method. Summary of the Invention [Means for solving the problem]

[0012] The present authors have discovered that Plin2 protein is an effective marker for nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) in circulating cells, and have surprisingly found that the expression level of this marker measured in hepatocytes is strongly and positively correlated with the expression level of this marker measured in white blood cells, particularly in peripheral blood polymorphonuclear leukocytes and monocytes, of the same individual.Furthermore, the present authors have also discovered that the expression level of Plin2 protein in blood cells and hepatocytes is also 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 in the description of the present invention).

[0013] The authors of the present invention have therefore devised a method for diagnosing NAFLD and / or NASH, which is carried out on a blood sample of an individual or on white blood cells extracted from said sample, in which the diagnosis of NAFLD and / or NASH must be considered confirmed if the Plin2 concentration in said sample is greater than that measured in one or more control samples taken from healthy individuals.

[0014] Measurement of Plin2 expression can further be advantageously used to monitor the effectiveness of treatment, since it correlates with the onset and severity of the disease. This allows the effectiveness of treatment to be assessed by comparing measurements of an individual's Plin2 blood concentration performed at a time t0, the start of monitoring the effectiveness of treatment, by comparing the value obtained in this measurement with values ​​obtained in one or more subsequent measurements performed at times tn. A decrease in Plin2 concentration over time represents therapeutic efficacy, where n is an integer greater than 0 and the times are sequential (following or next to) each other and all follow t0. A constant value over time indicates containment of the disease, while an increase in the value over time indicates therapeutic ineffectiveness.

[0015] Furthermore, subsequent measurements as described above allow for monitoring the progress of the disease even in the absence of pharmacological treatment, for example, after changes in the patient's diet and lifestyle, medications or weight loss surgery.

[0016] Therefore, the present invention advantageously makes it possible to diagnose and / or monitor NAFLD or NASH without the need for liver biopsy, with clear and obvious medical and economic advantages. Furthermore, the simplicity of the diagnosis and / or monitoring according to the present invention makes it possible to significantly expand the number of individuals who can be analyzed, making it easy to conduct tests on children, and even to confirm the health status of NAFLD or NASH in an entire population of patients who would not normally undergo liver biopsy. Furthermore, the analysis according to the present invention also makes it possible to conduct population screening.

[0017] Furthermore, given that the high incidence of cardiovascular damage in patients with NAFLD and NASH has been found to be independent of the presence or absence of other risk factors, early diagnosis of the disease could allow for preventive cardiovascular monitoring in these patients.

[0018] The authors of the present invention have discovered that, as mentioned above, Plin2 protein concentration values ​​correlate with the NAS score and also with the severity of NASH, which is defined herein 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 of NASH in a patient suffering from said pathology based on Plin2 protein concentration values ​​in said patient's biological sample as defined in the present invention.

[0019] The authors of the present invention have surprisingly also discovered that in the same biological samples, Pnpla3 and Rab14 protein expression levels allow the diagnosis of the presence of liver fibrosis and their severity.

[0020] The method of the invention can also be carried out using simple devices that allow the above-mentioned measurements (measurements) and optionally processing of the data obtained.

[0021] Therefore, the object of the present invention is to 1. A method for diagnosing non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), comprising: a. measuring the concentration of Plin2 protein in a blood sample of an individual or in a sample of white blood cells extracted from said blood sample; comparing the values ​​obtained at time point a with the concentration values ​​of Plin2 protein in a blood sample of a healthy individual and in a sample of white blood cells extracted from said blood sample; diagnosing NAFLD and / or NASH when the value measured at ca is greater than the value measured at b; 1. A method for diagnosing NAFLD or NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NAFLD and healthy patients, and identifying a cutoff value C0 of said concentration between patients with NAFLD and healthy patients; and measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NASH and healthy patients, and identifying a cutoff value C1 of said concentration between patients with NASH and healthy patients; comparing the value obtained at time point ca with a cut-off value obtained at time point b; diagnosing NAFLD when the value obtained in da is equal to or greater than the value C0 and smaller than the value C1, or diagnosing NASH when the value obtained in a is equal to or greater than the value C1; 1. A method for diagnosing NAS in a patient suffering from NAFLD or NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NAFLD or NASH and healthy patients, wherein in the population, including samples from patients with NAFLD, NAS values ​​are histologically defined in the patients as NAS1, NAS2, and NAS3 based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; The cutoff value C2 of the concentration between healthy patients and patients with NAFLD who have NAS1; Identifying a cutoff value C3 of the concentration between patients suffering from NAFLD and having NAS1 and patients suffering from NAFLD and having NAS2, and a cutoff value C4 of the concentration between patients suffering from NAFLD and having NAS2 and patients suffering from NASH and having NAS3; comparing the value obtained at time point ca with a cut-off value obtained at time point b; d.NAS degree, When the value obtained in a is equal to or greater than the cut-off value C2 and equal to or less than the cut-off value C3, it is determined as NAS1. When the value obtained in a is greater than the cutoff value C2 and less than or equal to the cutoff value C4, it is set as NAS2. a) diagnosing the patient as NAS3 when the value obtained in a) is greater than the cutoff value C4; 1. A method for diagnosing the severity of NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NASH and healthy patients, wherein the severity of the condition in the population, including samples from patients with NASH, is histologically defined in the patients as mild, moderate, or severe based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; The cutoff value C5 of the concentration between healthy patients and patients with mild NASH; The cutoff value of the concentration C6 between patients with mild NASH and patients with moderate NASH, Identifying a cutoff value C7 concentration between patients with moderate NASH and patients with severe NASH; comparing the value obtained at time point ca with a cut-off value obtained at time point b; d. The severity of NASH is assessed by When the value obtained in a is greater than the value C5 and less than the value C6, the condition is mild. If the value obtained in a is greater than the value C6 and less than the value C7, the condition is moderate. a) diagnosing the condition as severe when the value obtained in a) is greater than the value C7; 1. A method for diagnosing NAFLD or NASH, comprising: a. measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and which specifically binds to Plin2 and not to other proteins; comparing the value obtained at time point b with a cut-off value for that concentration, expressed in terms of mean fluorescence intensity (MFI), equal to 2.7 MFI and a cut-off value for that concentration equal to 1.0 MFI; diagnosing NAFLD when the value obtained in da is 1.0 MFI or more and 2.7 MFI or less, or diagnosing NASH when the value obtained in a is greater than 2.7 MFI; 1. A method for diagnosing NAS in a patient suffering from NAFLD or NASH, comprising: a. measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and which specifically binds to Plin2 and not to other proteins; comparing the value obtained at time point ca with cut-off values ​​for that concentration, expressed in terms of mean fluorescence intensity (MFI), equal to 1.0 MFI, 1.4 MFI and 2.7 MFI; diagnosing NAS equal to 1 when the value obtained in da is equal to or greater than the cut-off value of 1.0 MFI and equal to or less than the cut-off value of 1.4 MFI, diagnosing NAS equal to 2 when the value obtained in a is greater than the cut-off value of 1.4 MFI, or diagnosing NAS equal to 3 when the value obtained in a is greater than the cut-off value of 2.7 MFI, 1. A method for diagnosing the severity of NASH, comprising: a. measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and which specifically binds to Plin2 and not to other proteins; comparing the values ​​obtained at time point ca with cut-off values ​​for that concentration, expressed in terms of mean fluorescence intensity (MFI), equal to 2.7 MFI, 4 MFI and 6.3 MFI; diagnosing mild NASH when the value obtained by da is greater than the cutoff value of 2.7 MFI and equal to or less than the cutoff value of 4 MFI, diagnosing moderate NASH when the value obtained by a is greater than the cutoff value of 4 MFI and equal to or less than 6.3 MFI, or diagnosing severe NASH when the value obtained by a is greater than the cutoff value of 6.3 MFI, The method further comprises measuring protein concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample at a time point or in a sample of white blood cells extracted from the blood sample for diagnosing NASH, and comparing the protein concentration values ​​to those in blood samples from healthy patients and, optionally, patients with histologically defined grades of liver fibrosis for diagnosing liver fibrosis. 1. A method for monitoring the effectiveness of a therapeutic treatment of NAFLD or NASH in a patient, comprising: a. measuring the concentration value of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a sample of white blood cells extracted from said blood sample at the moment t0 when said monitoring is carried out; b. measuring a concentration value of Plin2 protein in a blood sample or a sample of white blood cells extracted from said blood sample of an individual suffering from NAFLD or NASH at one or more moments tn, where n is an integer greater than 0 and each tn corresponds to a moment after t0; a decrease in the concentration of Plin2 protein at one or more of said moments indicates the effectiveness of said therapeutic treatment; 1. A method for monitoring the progression of NAFLD or NASH in a patient, comprising: a. measuring the concentration value of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a sample of white blood cells extracted from said blood sample at the moment t0 when said monitoring is carried out; b. measuring a concentration value of Plin2 protein in a blood sample or a sample of white blood cells extracted from said blood sample of an individual suffering from NAFLD or NASH at one or more instants tn, where n is an integer greater than 0 and each tn corresponds to an instant after t; a decrease in the concentration of Plin2 protein at one or more of the instants indicates an improvement in NAFLD or NASH, while an increase in the concentration of Plin2 protein at one or more instants tn indicates a worsening of NAFLD or NASH; a computer program facilitating the implementation of said method, and a device for the automated determination of Plin2 protein concentration values ​​in blood samples, generally comprising: - a means for isolating PBMCs from a blood sample; -Means for measuring the concentration of Plin2 protein in the cytoplasm of said PBMCs; a control unit connected to said isolating means and said measuring means, Controlling and synchronizing their actuation in an automatic mode according to predetermined operating parameters; The method includes a device including a control unit that is programmed to automatically compare data on Plin2 protein concentration in the cytoplasm of the PBMC with reference data on Plin2 protein concentration.

[0022] term The term white blood cells for the purposes of the present invention has the meaning commonly known in the literature and includes different cell types such as neutrophils, eosinophils or acidophils, basophils, lymphocytes, granulocytes (or polymorphonuclear cells) subdivided into monocytes. For the purposes of the present invention, the term Plin2 refers to the human protein adipose differentiation-associated protein, also known as ADFP, ADRP or perilipin 2, which is encoded by the human gene ADFP, which maps to 9p22.1. NAFLD Non-alcoholic fatty liver disease NASH (non-alcoholic steatohepatitis) Pnpla3 refers to the human Pnpla3 protein, which is also known as adiponutrin, ADPN, acylglycerol O-acyltransferase, C22orf20, IPLA2 epsilon, DJ796I17.1, IPLA(2) epsilon, IPLA2-epsilon. Rab14 refers to the human Rab14 protein, also known as Rab-14, a Ras-related protein. [Brief explanation of the drawings]

[0023] [Figure 1] Panel A: Lipid droplets (light gray dots) in monocytes and hepatocytes; large, slightly more intensely stained nuclei are visible. Panel B shows Western blots of Plin2 in monocytes and liver. β-actin shows how the amount of protein detected in monocytes is similar to that detected in hepatocytes. [Figure 2] ORO staining in liver sections and monocytes from similar subjects is reported. [Figure 3] Bland-Altman graphs of the difference (Y-axis) and mean (X-axis) of Plin2 measurements by Western blot in monocytes and liver are reported. The two dotted lines indicate the limits of agreement. The difference between the values ​​obtained in the two measurements is reported on the Y-axis, while the mean is on the Y-axis. The dotted line reports the 95% boundary line (limit) of agreement between the two measurements. The graph shows that all points are within the limits of agreement, demonstrating a direct correlation between Plin2 expression in the liver and in peripheral blood cells. [Figure 4] 1 is an exemplary block diagram of a preferred embodiment of a device according to the present invention; [Figure 5] 10 is an exemplary illustration of the use of a further preferred embodiment of the device according to the present invention; [Figure 6] 10 is an exemplary illustration of the use of a further preferred embodiment of the device according to the present invention; [Figure 7] Correlation between mean Plin2 (MFI) level and NAS stage, where the independent variable on the X-axis is the mean value of Plin2, while the dependent variable is NAS stage. [Figure 8] NASH normalized importance of Plin2. [Figure 9] Fibril-normalizing significance of Pnpla3 and Rab14. [Figure 10] Fibrosis-normalized importance of Plin2. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present authors have surprisingly discovered that Plin2 protein is a marker for NAFLD and NASH, and that the protein concentration in blood cells (white blood cells) correlates with the expression of this protein in liver cells, and that the concentration is also proportional to the NAS score and the severity of NASH disease.The present authors have therefore discovered that by analyzing blood samples without the need to rely on liver biopsy, it is possible to use measurement of the concentration of Plin2 protein to perform the diagnosis of NAFLD or NASH, to diagnose the severity of NAFLD or NASH as defined in this document, such as mild, moderate, or severe, or to monitor the effectiveness of therapeutic treatment for NAFLD or NASH, or to monitor the course of NAFLD or NASH over time.

[0025] Therefore, the present invention provides a. measuring the concentration of Plin2 protein in a blood sample of an individual or in a sample of white blood cells extracted from said blood sample; comparing the values ​​obtained at time point a with the concentration values ​​of Plin2 protein in a blood sample of a healthy individual and in a sample of white blood cells extracted from said blood sample; Provided is a method for diagnosing non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), comprising the step of diagnosing NAFLD and / or NASH when the value measured by ca is greater than the value measured by b.

[0026] In one embodiment, in step a, a blood sample is used which may be processed for the purpose of isolating white blood cells from the blood sample by techniques known to those skilled in the art.

[0027] The method thus comprises a step preceding the measurement of the concentration of Plin2 protein in a sample, the blood sample being subjected to a treatment to isolate the white blood cells contained therein. In one embodiment, the method may comprise a step of isolating polymorphonuclear cells (PMNs) and / or monocytes from the blood sample to be analyzed or the white blood cells isolated therefrom.

[0028] Any protocol known in the literature for current use for isolating white blood cells from a blood sample can be used.

[0029] By way of example only, polymorphonuclear cells can be isolated from blood samples as follows: Blood is drawn into a test tube containing EDTA (final concentration 4 mM) to prevent clotting. One or more discontinuous Percoll components (consisting of colloidal silica particles (23% w / w in water) with a diameter of 15-30 nm, coated with polyvinylpyrrolidone (PVP)) are then placed in the tube, and 15 ml of 62% Percoll is gently layered underneath with 15 ml of 75% Percoll using a syringe with a thin catheter, avoiding mixing of the two suspensions.

[0030] Eight to ten milliliters of blood was then layered onto the components described above, and the tubes were centrifuged for a total of 25 minutes at 20°C, including 10 minutes at 200 rpm and then 15 minutes at 400 rpm, thus separating the blood's geometric components based on density and size: red blood cells and most eosinophils settled to the bottom of the tube, polymorphonuclear cells settled at the interface between the two Percoll suspensions, and lymphomonocytes located between 62% of the Percoll and the plasma.

[0031] After discarding the plasma and lymphomonocytes, the polymorphonuclear cell-containing band was collected using a glass Pasteur pipette, collected in a test tube, and washed with HEPES / BSA by centrifugation at 250 rpm for 7 minutes at 20°C. The resulting bottom layer was rapidly lysed to discard any red blood cells that may be present and resuspended in 3 parts hypotonic solution. After approximately 15 seconds of vortexing, the tonicity of the medium was restored by adding 7 parts isotonic solution.

[0032] After a further wash at 250 rpm for 7 min at 20°C, the cell pellet was resuspended and kept in a known volume of HEPES / BSA until use. Neutrophil concentrations were measured using an electronic particle counter or under a light microscope using a hemocytometer.

[0033] By way of example only, various types of monocytes can be isolated from blood samples by techniques such as the use of CD14-specific recombinant Fab fragments of microbiologically expressed monoclonal antibodies against surface cell markers, and commercially available kits are available for this purpose, such as the IBA GmbH™ CD14 isolation kit for FABian™.

[0034] Measurement of Plin2 protein concentration can be performed according to any method available to those skilled in the art. Merely by way of example, in the methods described herein, measurement can be performed by Western blot, cytofluorometry, ELISA, immunofluorescence, quantitative PCR, enzyme-linked immunospot assay, or localized surface plasmon resonance fiber-tip probe system, or by using a device prepared for such measurement as described below. All of the methods described above are known to those skilled in the art, who know which sample to perform the test on and which proteins to quantify, can select the protocol they deem most appropriate, and can use the most suitable reagents available on the market. All reagents required to detect and quantify Plin2 in a sample according to the present specification are known and commercially available. Therefore, once the method of the present invention is publicly known, it does not require any special ingenuity on the part of those skilled in the art.

[0035] By way of example, the techniques listed above are summarized and for each step the steps commonly used by those skilled in the art are shown.

[0036] Western blotting allows monitoring protein expression in cells, whereby the presence, amount and molecular weight of a particular antigen can be determined in three ways: 1) Protein extraction and dosage; 2) separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); 3) Western blotting: transferring the proteins separated from the gel onto a nitrocellulose membrane (blot), exposing the membrane to an antibody directed against the protein of interest (primary antibody), exposing the membrane to an antibody (secondary antibody) directed against the species used as the primary antibody, and developing the membrane with chemiluminescence (ECL); 4) Image densitometry analysis.

[0037] Cytofluorometry is a laboratory technique that can detect, identify and enumerate specific cells or proteins contained therein.

[0038] Cytofluorimetric techniques anticipate various stages.

[0039] Suspension of cell samples in fluid Before testing, the sample is treated with specific dyes that are capable of distinguishing cell subtypes, depending on the cells being analyzed. These dyes, fluorescent dyes, bind to monoclonal antibodies directed against specific cell regions or marker antigens.

[0040] The sample containing the cells to be labeled is introduced into an instrument called a cytofluorometer.

[0041] In this device, the cell-containing fluid is forced into a flow chamber and then through a very narrow orifice to generate a stream of cells organized in a single file inside. This stream of cells is then placed in front of a detector, which can analyze each cell present in the stream at very high speeds (hundreds to thousands of cells per second).

[0042] A cytofluorometer contains one or more lasers and multiple detectors capable of identifying some characteristic feature of each cell. Each laser interacts with the cells present in the stream, producing a characteristic scattering effect for each cell depending on its characteristics. This characteristic can be physical (cell size and complexity) or can vary depending on the signal generated by the laser-intercepting dye (fluorochrome). This combination of information creates a characteristic profile of each cell present in the sample.

[0043] The signal detected by one or more detectors (or a single detector) is amplified (by a photomultiplier tube) and sent to a computer, where it is converted to digital form and displayed on the computer or printed.

[0044] The data is presented in graphical form.

[0045] ELISA (enzyme-linked immunosorbent assay) is a very frequently used technique that is based on the chemical conjugation of an enzyme to an antibody or antigen. The activity of these enzymes can be easily monitored, allowing for accurate quantification of the conjugate complex concentration. Depending on the particular method used, ELISA can be used to administer either antigens or antibodies.

[0046] antigen recognized by a specific antibody (immunity) Analyte (antigen or antibody) adsorbed on the system (adsorbent) surface An antigen / antibody can be recognized by a (second) enzyme-linked antibody, causing a reaction whose product is stained.

[0047] Quantitative PCR, or real-time PCR, is a technique used to quantify nucleic acids (in this case, mRNA-encoded Plin2 protein) by measuring the fluorescence emitted by a fluorophore. This technique couples amplification and quantification within a single reaction. In real-time PCR reactions, fluorescence increases proportionally to the accumulation of PCR products. Those skilled in the art would have no problem designing suitable probes for performing RT-PCR, since the gene and DNA encoding Plin2 protein are publicly known in the literature. Enzyme-linked immunosorbent spot assay (ELISPOT) analysis of PBMC samples is based on a limited-time cell incubation in a 96-well plate pre-functionalized (coated) by adsorption of a monoclonal antibody with high affinity against the cytokine of interest produced during incubation. The combination of a biotinylated anti-reference protein antibody and an anti-biotin enzyme-linked secondary antibody precipitates the reaction product by forming a spot (dye accumulation), allowing detection of protein production within a defined area in the presence of a chromogenic substrate for the enzyme. The technique can be added to the PBMC contents after cell lysis.

[0048] Surface plasmons have been 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, such as in proteins. Technically, the angle of minimum reflection (maximum absorption) is measured.

[0049] Any analytical technique known in the literature that is suitable for quantifying the Plin2 concentration in a blood sample or cell sample as defined above can be used in any method of the invention.

[0050] Furthermore, a device according to the invention may be designed to perform quantification of Plin2 protein in a sample of interest by one or more of the detection techniques described above.

[0051] In a particular embodiment, the measurement can be carried out by cytofluorimetry using as a protein marker an antibody specific for the Plin2 protein (or a derivative or fragment thereof as defined above), i.e. an antibody that binds only to the Plin2 protein, labeled with a suitable fluorescent dye.

[0052] In one embodiment, the fluorescent dye may be any one that is detectable by a commercially available cytofluorometric analyzer, such as, for example, a fluorescent dye having the following characteristics: [Table 1]

[0053] In one embodiment, Alexa Fluor 488 fluorescent dye (Invitrogen) can be used, which has the following technical features and can be commonly used as an alternative to FITC or Cy2:

[0054] Analysis of fluorescence intensity and mean fluorescence intensity (MFI) values ​​can be calculated by suitable software that is publicly available and can be assessed by use of commercially available cytofluorometers.

[0055] Thus, according to one embodiment, the fluorescent dye used may be Alexa Fluor 488 fluorescent dye having the technical characteristics defined above, and the data may be analyzed using an FC 500 cytofluorimeter (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA), which has the technical characteristics of that product at the time of filing this application.

[0056] Those skilled in the art will understand in any way how to adapt the cutoffs expressed in terms of MFI provided herein for comparative assays calculated using Alexa Fluor 488 fluorochrome or fluorochromes having the characteristics reported in the table above, using an FC 500 cytofluorimeter (Beckman Coulter, Brea, CA) or a cytofluorometer with similar technical characteristics, and Kaluza software (Beckman Coulter, Brea, CA), with cutoffs expressed in terms of MFI calculated using other fluorochromes and other devices and software.

[0057] MFI is simply the arithmetic mean.

[0058] According to the present invention, the cytofluorometer preferably has the technical characteristics of the cytofluorometers set out above and which are publicly available at the time of filing this application.

[0059] All of the process embodiments described above are applicable to any of the methods provided herein.

[0060] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: 1. A method for diagnosing NAFLD or NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NAFLD and healthy patients, and identifying a cutoff value C0 of said concentration between patients with NAFLD and healthy patients; and measuring the concentration values ​​of Plin2 protein in a population of blood samples from patients with NASH and healthy patients, and identifying a cutoff value C1 of said concentration between patients with NASH and healthy patients; comparing the value obtained at time point ca with a cut-off value obtained at time point b; The method includes a step of diagnosing NAFLD when the value obtained in da is equal to or greater than the value C0 and smaller than the value C1, or diagnosing NASH when the value obtained in a is equal to or greater than the value C1.

[0061] The present invention also provides a method for diagnosing the NAS score of a patient suffering from NAFLD or NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NAFLD or NASH and healthy patients, wherein in the population including samples from patients with NAFLD, NAS score values ​​are histologically defined in the patients as NAS1, NAS2, and NAS3 based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; The cutoff value C2 of the concentration between healthy patients and patients suffering from NAFLD with an NAS score of 1; Identifying a cutoff value C3 of the concentration between patients suffering from NAFLD with NAS1 and patients suffering from NAFLD with NAS score 2, and a cutoff value C4 of the concentration between patients suffering from NAFLD with NAS2 and patients suffering from NASH with NAS score 3; comparing the value obtained at time point ca with a cut-off value obtained at time point b; d. NAS score, When the value obtained in a is equal to or greater than the cutoff value C2 and equal to or less than the cutoff value C3, the NAS score is 1. When the value obtained in a is greater than the cutoff value C2 and less than or equal to the cutoff value C4, the NAS score is 2. When the value obtained in step a is greater than the cutoff value C4, the patient is diagnosed with an NAS score of 3.

[0062] The NAS score according to the present invention is as defined in the literature and is typically assigned according to the following parameters: [Table 2]

[0063] The present invention also provides a method for diagnosing the severity of NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NASH and healthy patients, wherein the severity of the condition in the population, including samples from patients with NASH, is histologically defined in the patients as mild, moderate, or severe based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; The cutoff value C5 of the concentration between healthy patients and patients with mild NASH; The cutoff value of the concentration C6 between patients with mild NASH and patients with moderate NASH, Identifying a cutoff value C7 concentration between patients suffering from moderate NASH and patients suffering from severe NASH; comparing the value obtained at time point ca with a cut-off value obtained at time point b; d. The severity of NASH is assessed by When the value obtained by a is greater than the value C5 and less than the value C6, the condition is mild. If the value obtained by a is greater than the value C6 and less than the value C7, the condition is moderate. a) is greater than the value C7, the method comprising the step of diagnosing the condition as severe.

[0064] Therefore, according to the present specification, the cut-off value of Plin2 concentration is The cutoff C0 between patients with NAFLD and healthy patients, The cutoff C1 between patients with NASH and healthy patients, The cutoff C2 between healthy patients and patients with NAFLD who have NAS1; The cutoff C3 between patients with NAS1 and NAFLD and patients with NAS2 and NAFLD, The cutoff C4 between patients with NAS2, suffering from NAFLD, and patients with NAS3, suffering from NASH, The cutoff C5 between healthy patients and patients with mild NASH, The cutoff C6 between patients with mild NASH and those with moderate NASH, The cutoff between patients with moderate and severe NASH is C7.

[0065] The definition of disease severity according to the present invention is based on histological analysis according to the NAFLD Activity Score (NAS), which is commonly used in the literature (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 histological score based on the presence of steatosis, lobular and portal inflammation, and puffy hepatocyte degeneration ("puffiness") in liver biopsies, and is widely used and validated by international liver societies. Each component can be grade 1 (mild), grade 2 (moderate), or grade 3 (severe).

[0066] [Table 3]

[0067] In fact, the authors of the present invention surprisingly found that the Plin2 protein concentration values ​​of analyzed blood samples from patients with known NASH severity correlate with said grade, and that elevated Plin2 protein concentrations are directly proportional to the severity of the condition.

[0068] In a preferred embodiment, the method described above uses cytofluorometric techniques to assess the expression of Plin2 protein, and is carried out by using a monoclonal antibody labeled with a suitable fluorescent dye that specifically binds to Plin2 and not to other proteins, the cutoff being expressed in terms of mean fluorescence intensity (MFI).

[0069] In a more preferred embodiment, this is by using the fluorescent dyes, software and devices described above, or the fluorescent dyes described above plus the software and devices that are the subject of the present invention as defined below and in the claims. According to the literature and the present invention, NASH necroinflammatory grades are classified into grade 1 (mild), grade 2 (moderate), and grade 3 (severe) based on the grade of hepatocellular steatosis, swelling and damage, and (intralobular and portal) inflammation (table above).

[0070] The present authors have demonstrated that certain cutoffs (expressed herein in terms of MFI with the fluorochromes, software and devices defined below) applicable to the methods described above can be computerized (see the Examples section).

[0071] Therefore, the object of the present invention is also to 1. A method for diagnosing NAFLD or NASH, comprising: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said concentration being measured by cytofluorometry using a monoclonal antibody that is labeled with a suitable fluorescent dye and that specifically binds to Plin2 and not to other proteins; comparing the value obtained at time point b with a cut-off value for that concentration, expressed in terms of mean fluorescence intensity (MFI), equal to 2.7 MFI and a cut-off value for that concentration equal to 1.0 MFI; The method includes a step of diagnosing NAFLD when the value obtained in da is 1.0 MFI or more and 2.7 MFI or less, or a step of diagnosing NASH when the value obtained in a is greater than 2.7 MFI.

[0072] The method for diagnosing the NAS score of a patient suffering from NAFLD or NASH comprises: a. measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and which specifically binds to Plin2 and not to other proteins; comparing the value obtained at time point ca with cut-off values ​​for that concentration, expressed in terms of mean fluorescence intensity (MFI), equal to 1.0 MFI, 1.4 MFI and 2.7 MFI; a step of diagnosing the patient as having an NAS score of 1 when the value obtained by da is equal to or greater than the cutoff value of 1.0 MFI and equal to or less than the cutoff value of 1.4 MFI, as having an NAS score of 2 when the value obtained by a is greater than the cutoff value of 1.4 MFI, or as having an NAS score of 3 when the value obtained by a is greater than the cutoff value of 2.7 MFI, Methods for NASH severity include: a. measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said concentration being measured by cytofluorometry using a monoclonal antibody that is labeled with a suitable fluorescent dye and that specifically binds to Plin2 and not to other proteins; comparing the values ​​obtained at time points ca with cut-off values ​​for the concentrations expressed in terms of mean fluorescence intensity (MFI) equal to 2.7 MFI, 4 MFI and 6.3 MFI; The method includes a step of diagnosing mild NASH when the value obtained by da is greater than the cutoff value of 2.7 MFI and equal to or less than the cutoff value of 4 MFI, moderate NASH when the value obtained by a is greater than the cutoff value of 4 MFI and equal to or less than 6.3 MFI, or severe NASH when the value obtained by a is greater than the cutoff value of 6.3 MFI.

[0073] Such a method is In one embodiment, Alexa Fluor 488 fluorescent dye, or a fluorescent dye having the characteristics reported in the table above, is used with an FC 500 cytofluorimeter (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA). Similarly, different cutoffs C0 to C7 can be defined by using a Plin2 protein detection and quantification system.

[0074] As indicated above, the authors also surprisingly discovered that assessment of the concentrations of Pnpla3 and Rab14 proteins in a blood sample at a time point for diagnosing NASH, or in a sample of white blood cells extracted from said blood sample, makes it possible to define whether liver fibrosis is present or absent and its severity.

[0075] In the present invention, for the purpose of defining liver fibrosis, the same definition as that used in the literature (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) is used, based on the location and extent of fibrosis, and the following scoring system is used to determine the stage: Stage 1, perisinusoidal fibrosis in zone 3; Stage 2, portal fibrosis with the above-mentioned Stage 1; Stage 3, Stage 2 plus bridging fibrosis; and Stage 4, cirrhosis. The NASH Clinical Research Network (NASH CRN) then subdivides Stage 1 into three categories: Stage 1A, mild perisinusoidal fibrosis in zone 3; Stage 1B, moderate periportal fibrosis in zone 3; and Stage 1C, portal / periportal fibrosis only. Stage 1C fibrosis is occasionally observed in children or severely obese patients.

[0076] [Table 4]

[0077] Therefore, the present invention also provides a further step applied to the method reported above. Once NASH is diagnosed from a patient's blood sample, it is also possible to diagnose the presence or absence of fibrosis and its pathological stage from the same sample or from a further blood sample from the patient.

[0078] The process of diagnosing fibrosis staging can also be performed on blood samples from patients suffering from NASH as defined herein and in the claims, and the diagnosis of NASH is performed in a different manner than described and claimed in the present invention.

[0079] Thus, in any of the embodiments described herein, step e can be replaced by step e' of measuring concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample of a patient diagnosed with NASH or in a sample of white blood cells extracted from that blood sample, and step g can therefore be replaced by step g' of comparing the value obtained at time e' with the cut-off value obtained at time f.

[0080] Therefore, an object of the present invention is any one of methods for diagnosing NASH alone and / or its severity, e. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample or a sample of white blood cells extracted from the blood sample at time point a for diagnosing NASH; f. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a group of blood samples from patients with liver fibrosis and healthy patients, and identifying the cutoff value C8 of the concentration between patients with liver fibrosis and healthy patients; comparing the value obtained at time ge with the cut-off value obtained at time f; and diagnosing liver fibrosis when the value obtained at time point he is greater than the value C8, or comprising steps e', f, g' and h. The object of the present invention is also any one of the methods for diagnosing only NASH and / or its severity, e. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample at time point a or in a sample of white blood cells extracted from the blood sample to diagnose NASH; f. measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a group of blood samples from patients with liver fibrosis and healthy patients, and systematically defining the severity of the condition as stage 1, stage 2, or stage 3 based on the location and extent of liver fibrosis; The cutoff value of the concentration C9 between healthy patients and patients with stage 1 liver fibrosis, the cutoff value C10 of the concentration between patients with stage 1 liver fibrosis and patients with stage 2 liver fibrosis; Identifying a cut-off value C11 of the concentration between patients suffering from stage 2 liver fibrosis and patients suffering from stage 3 liver fibrosis; comparing the value obtained at time ge with the cut-off value obtained at time f; h. The stage of liver fibrosis is determined by: If the value obtained at time e is equal to or greater than the value C9 and smaller than the value C10, then stage 1 is reached. If the value obtained at time e is equal to or greater than the value C10 and equal to or less than the value C11, it is stage 2. diagnosing stage 3 when the value obtained at time e is greater than the value C11, or comprising steps e', f, g' and h', wherein all references to time e in step h above are changed, mutatis mutandis, to references to time e'.

[0081] The object of the present invention is also a method for diagnosing liver fibrosis and for diagnosing the stage of liver fibrosis in a blood sample or white blood cells from a patient diagnosed with NASH, comprising the steps described above, wherein steps e and g are replaced by steps e' and g'.

[0082] In summary, the cut-off values ​​for Pnpla3 and Rab14 concentrations defined herein are: The cutoff C8 between patients with liver fibrosis and healthy patients, The cutoff C9 between healthy patients and patients with stage 1 liver fibrosis, The cutoff C10 between patients with stage 1 liver fibrosis and patients with stage 2 liver fibrosis, The cutoff C11 is between patients with stage 1 liver fibrosis and patients with stage 3 liver fibrosis.

[0083] The concentration values ​​of the above-mentioned proteins can be measured by Western blot, or cytofluorimetric analysis, or ELISA, or quantitative PCR.

[0084] In a specific embodiment, this concentration value is measured by cytofluorometry using a monoclonal antibody labeled with a suitable fluorescent dye that specifically binds to Pnpla3 and not to other proteins, and a monoclonal antibody that specifically binds to Rab14 and not to other proteins, and the cutoff is expressed in terms of mean fluorescence intensity (MFI).

[0085] As mentioned above, concentration values ​​of Pnpla3 and Rab14 proteins can be calculated by using all the described embodiments and devices that calculate Plin2 concentration values, for example fluorescent dyes with the following characteristics: [Table 5]

[0086] Thus, according to one embodiment, the fluorescent dye used may be Alexa Fluor 488 fluorescent dye having the technical characteristics defined above, and the data may be analyzed using an FC 500 cytofluorimeter (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA) having the technical characteristics of that product publicly available at the time of filing this application.

[0087] The present authors have demonstrated that certain cutoffs (expressed herein in terms of MFI with the fluorochromes, software and devices defined below) applicable to the methods described above can be computerized (see the Examples section).

[0088] Therefore, the object of the present invention is also any one of the methods for diagnosing NASH alone and / or its severity, measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample at the time of ea or at the time of diagnosing NASH, or in a sample of white blood cells extracted from said blood sample, said values ​​being measured by cytofluorometry using a monoclonal antibody that is labeled with a suitable fluorescent dye and that specifically binds to PNPLA but not to other proteins, and a monoclonal antibody that specifically binds to Rab14 but not to other proteins; comparing the value obtained at the ge time point with a cut-off value, expressed in terms of mean fluorescence intensity (MFI), of 1.24 MFI or greater; diagnosing liver fibrosis when the value obtained at time point is 1.24 MFI or greater; and any one of the methods for diagnosing only NASH and / or its severity, measuring the concentration values ​​of Pnpla3 and Rab14 proteins in a blood sample at the time of ea or at the time of diagnosing NASH, or in a sample of white blood cells extracted from said blood sample, said values ​​being measured by cytofluorometry using a monoclonal antibody that binds specifically to PNPLA but not to other proteins, and a monoclonal antibody that binds specifically to Rab14 but not to other proteins, labeled with a suitable fluorescent dye, said cut-off being expressed in terms of mean fluorescence intensity (MFI); comparing the value obtained at time ga with cut-off values ​​for said concentrations expressed in terms of mean fluorescence intensity (MFI) equal to 1.24 MFI, 2.3 MFI and 3.10 MFI; The method further comprises diagnosing mild stage 1 liver fibrosis when the value obtained at time point he is 1.24 MFI or more and less than 2.4 MFI, stage 2 liver fibrosis when the value obtained at time point e is 2.4 MFI or more and less than 3.10 MFI, and stage 3 liver fibrosis when the value obtained at time point e is 3.10 MFI or more.

[0089] Again, the object of the present invention is also a method for diagnosing liver fibrosis and for diagnosing the stage of liver fibrosis in a blood sample or white blood cells from a patient diagnosed with NASH, comprising the steps as described above, wherein steps e and g are replaced by steps e', g' and h', in which case, mutatis mutandis, all references to time point e in step h above are changed to references to time point e'.

[0090] The values ​​reported above are defined using Alexa Fluor 488 fluorescent dye, or a fluorescent dye with the characteristics reported in the table above, using a Cytofluometer FC 500 (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA). Obviously, other cutoff values ​​can be assigned by using different detection systems.

[0091] Those skilled in the art will understand how, for comparative assays, cutoffs calculated using other devices and software fit in any way to the cutoffs provided herein expressed in terms of MFI calculated using an FC 500 Cytofluometer (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter, Brea, CA) using Alexa Fluor 488 fluorescent dye or a fluorescent dye having the characteristics reported in the table above.

[0092] Furthermore, the present invention provides a method for monitoring the effectiveness of a therapeutic treatment of NAFLD or NASH in a patient, comprising: a. measuring the concentration value of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a sample of white blood cells extracted from said blood sample at the moment t0 when said monitoring is carried out; b. measuring a concentration value of Plin2 protein in a blood sample or a sample of white blood cells extracted from said blood sample of an individual suffering from NAFLD or NASH at one or more moments tn, where n is an integer greater than 0 and each tn corresponds to a moment after t0; wherein a decrease in the concentration of Plin2 protein at one or more of said moments indicates the effectiveness of said therapeutic treatment.

[0093] In carrying out the monitoring method described above, time t0 is the time at which monitoring begins, which may correspond to a moment before the beginning of treatment, or it may be the moment from which monitoring begins, which may also be when treatment begins.

[0094] The moments tn are moments that follow each other (next to each other) with increasing value n and that follow moment t0.

[0095] Thus, instant t0 is the instant at which monitoring begins, and the concentration value of Plin2 in the blood sample is considered the value at which evaluation of therapeutic effectiveness begins. Concentration measurements are then repeated at successive times (next to each other) following t0, proceeding from 1 onwards, where n is an integer greater than 0, such that instant t1 precedes instant t2 which precedes instant t3, and so on.

[0096] A detectable and significant decrease in Plin2 concentration in a patient's blood sample analyzed at times subsequent to t, relative to the value measured at time t, indicates the effectiveness of the therapeutic treatment in ameliorating NAFLD or NASH. Maintenance of a constant value over time indicates the effectiveness of the therapeutic treatment in treating NAFLD or NASH, while an increase in value over time relative to the value measured at time t indicates the ineffectiveness of the therapeutic treatment.

[0097] In some cases, dietary and lifestyle changes are suggested to patients before pharmacological treatment is administered, and in these cases it is useful to monitor the progression of the disease over time to see, for example, whether the dietary and lifestyle changes are positively affecting the disease.

[0098] Any method for monitoring the course of the disease, with or without evaluation of possible therapeutic efficacy, may be of interest to the treating physician. Therefore, the object of the present invention is also a method for monitoring the progression of NAFLD or NASH in a patient over time, comprising: a. measuring the concentration value of Plin2 protein in a blood sample of an individual suffering from NAFLD or NASH or in a sample of white blood cells extracted from said blood sample at the moment t0 when said monitoring is carried out; b. measuring a concentration value of Plin2 protein in a blood sample or a sample of white blood cells extracted from said blood sample of an individual suffering from NAFLD or NASH at one or more instants tn, where n is an integer greater than 0 and each tn corresponds to an instant after t; A decrease in the concentration of Plin2 protein at one or more of said moments indicates an improvement in NAFLD or NASH, while an increase in the concentration of Plin2 protein at one or more moments tn indicates a worsening of NAFLD or NASH, while a constant maintained value over time indicates a plateau in the disease.

[0099] What is said about the instants t0 and tn when describing the method for monitoring a therapeutic treatment applies to methods for monitoring in general.

[0100] All embodiments of the diagnostic method described below also apply to the monitoring method described below.

[0101] Generally, in carrying out the methods described herein, blood samples may also be taken from organs etc., although the use of peripheral blood is preferred.

[0102] In particular, in carrying out the methods described herein, it is preferable to measure the concentrations of Plin2, Pnpla3 and Rab14 in white blood cells, more particularly polymorphonuclear cells (PMNs) and / or monocytes, isolated from the peripheral blood.

[0103] Similar methods for monitoring efficacy and disease progression as shown above for NAFLD or NASH can be implemented, with necessary modifications, when monitoring treatment efficacy and progression of fatty liver, using as examples the concentration values ​​of the markers Pnpla3 and Rab14 in blood samples of patients suffering from fatty liver.

[0104] The present invention further provides a device for the automated measurement of Plin2 and / or Pnpla3 and / or Rab14 protein concentration values ​​in a blood sample taken from an individual, generally comprising: A means for isolating PBMCs from a blood sample; A means for measuring the concentration of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein in the cytoplasm of said PBMC; and a means for correlating the obtained measurement with a comparative value, which may be a value obtained from a healthy individual, or one or more values ​​of the concentration of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein in the cytoplasm of PBMCs from a blood sample of the same individual obtained previously.

[0105] As mentioned above, the device according to the present invention can be implemented to carry out measurements of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein in a blood sample taken from an individual by one or more of the detection techniques described above.

[0106] Depending on the technique chosen for the isolation and lysis of PBMCs, the above-mentioned procedures may be carried out according to various variants, some of which will be explained below. For example, in the case of a separation format using components that selectively bind to PBMCs or monocytes, the device may comprise: - A collection chamber that is easy to receive the blood sample; - mixing means contained within the collection chamber, configured to mix the blood sample with components susceptible to binding to polymorphonuclear leukocytes (PBMC) present in the sample and, if necessary, an anticoagulant; - a moving means configured to apply mechanical vibration and rotational stress to the mixture obtained by the mixing means and to transport the mixture to a filtering means; - said filtration means configured to retain said components bound to said polymorphonuclear corpuscles (PBMCs) and capable of eluting the remainder of said mixture; - means for isolating said polymorphonuclear corpuscles (PBMCs) from the components to which they are bound, said means being configured to favour separation between said polymorphonuclear corpuscles (PBMCs) and said components; - a means for treating the polymorphonuclear cells (PBMCs) isolated by said isolation means, the means being configured to allow binding of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein, when present, to a reagent specific for said protein, said reagent being fluorescent at one or more predetermined wavelengths; - at least one radiation source, configured to emit radiation, for example in the form of light waves, at a predetermined wavelength such that the reagent emits fluorescence; - means for acquiring an image of the irradiated sample; - a control unit (7) connected to all said means and said radiation source (12) and programmed to control and synchronize their activation in automatic mode according to predetermined operating parameters, said control unit further configured to process said images in order to quantify the Plin2 protein and / or Pnpla3 protein and / or Rab14 protein concentration present in said sample.

[0107] The control unit is further configured to compare the measured concentration with one or more reference data of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein concentration obtained from measurements performed on blood samples of healthy subjects and / or on blood samples of the same individual taken at different moments.

[0108] In one embodiment, for example with reference to Plin2 protein, the reference data for Plin2 protein concentration is data relating to healthy subjects, and the control unit 7 is further programmed to detect a non-alcoholic fatty liver disease (NAFLD) condition if the Plin2 protein concentration data obtained from sample analysis is greater than the reference data for Plin2 protein concentration.

[0109] Similar to those described above, the level of Pnpla3 and Rab14 protein concentrations can be used to detect liver fibrosis conditions and optionally assess their severity. According to a further embodiment, the reference data of Plin2 protein concentration are data related to the same patient and detected at a time (t0) prior to the time (tn) at which the blood sample is detected, and the control unit 7 is further programmed to detect the progression of the disease over time.

[0110] For example, if the Plin2 protein concentration data obtained from the sample analysis is greater than the reference data, an improvement in NAFLD is detected. If the Plin2 protein concentration data obtained from the sample analysis is smaller than the reference data, a worsening of NAFLD is detected, whereas if the Plin2 protein concentration data obtained from the sample analysis is equal to the reference data, a stagnation of the disease is detected.

[0111] Preferably, Plin2 protein concentration data obtained from sample analysis is considered to be equal to the reference data when the difference between the two data is between about 0% and about 5%.

[0112] A first preferred embodiment and a second preferred embodiment of the device are illustrated by way of example in FIGS.

[0113] 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 puncturing means 2 that facilitates puncturing the skin of an individual from whom the blood sample is to be collected. For example, the puncturing means 2 may be suitable for puncturing the patient's skin with a fingertip. The puncturing means particularly includes at least a protruding terminal element having a pointed end, e.g., a needle. The puncturing means 2 is supported on the exterior surface of the device 1 and is configured and articulatable to assume a minimally burdensome configuration when not in use, in which the pointed element faces the exterior surface of the device 1, and in the use configuration, the pointed element faces away from the exterior surface of the device 1. In a preferred embodiment, the puncturing means 2 includes a plurality of needles, such as micrometers in size, otherwise referred to as "microneedles." Usefully, the microneedles may be arranged relative to one another to form a matrix of pointed elements suitable for collecting a blood sample from a patient. Such arranged micrometer size allows multiple skin punctures to be performed to facilitate extraction of blood samples.

[0114] When the device according to the present invention directly comprises a puncturing means 2 for obtaining a blood sample, once the blood sample is obtained, it is collected in the above-mentioned means 2 and then transported to a collection chamber 3 in the device 1 by suitable fluid connection means or means for transporting 4. Such transport can occur by suction of the blood sample. In particular, the suction is performed by means for transporting 4, which have a respective suction port in the puncturing means 2. In particular, the means for transporting 4 can be of the silicone diaphragm pump type or of the vacuum micropump type. Preferably, the diaphragm pump has the following dimensions: - height equal to 0.6 mm, - width equal to 5 mm, - a length equal to 5 mm.

[0115] Diaphragm pumps are therefore small enough to be easily moved, and the device is fairly non-bulky, lightweight and easy to transport.

[0116] The means for conveying 4 may further comprise a collection duct or cannula, for example a rubber catheter, which may allow the sample to come into contact with an anticoagulant.

[0117] Alternatively, samples independently extracted by the devices described herein can be injected directly into a collection chamber, preferably after treatment with an anticoagulant.

[0118] In an alternative embodiment, the sample is introduced or transported to collection chamber 3 before contacting with the anticoagulant.

[0119] Preferably, the device 1 comprises means 14, 5, 6, 19 for isolating polymorphonuclear cells (PBMCs) from a blood sample.

[0120] The isolation means 14, 5, 6, 19 according to the present invention may preferably comprise a mixing means 14 inserted into the collection chamber 3 and configured to mix the blood sample with a component or group of components susceptible to isolating PBMCs (also defined herein as "PBMC isolation means"). For example, the group of components may comprise components present in the sample susceptible to binding to polymorphonuclear cells (PBMCs), and may be accompanied by an anticoagulant if necessary (i.e. when the blood sample has not yet been treated with an anticoagulant).

[0121] In the implementation of the devices described herein, the components may be represented, for example, by suitable microbeads (e.g., PluriBead® or Dynabeads® type technology) onto which are coupled antibodies against PBMC-specific antigens that bind to CD3+ T and thus PBMCs.

[0122] The device 1 may further comprise a first container s1 and / or a second container s2 holding an anticoagulant and a component capable of binding to polymorphonuclear cells (polymorphonuclear leukocytes), respectively. Each container s1, s2 preferably comprises a respective delivery means 15.

[0123] Additionally, the isolation means 14, 5, 6, 19 may include a transfer means 5 and a filtering means 6, which are described below.

[0124] By mixing, the PBMCs bind to the above-mentioned components. The resulting mixture is subjected to mechanical vibration and / or rotational stress, preferably caused by the action of dedicated moving means 5 contained inside the collection chamber 3. In particular, the moving means 5 may be implemented by at least one rotating and / or vibrating conveyor belt, which conveys the thus obtained mixture upward.

[0125] The stressing of the mixture by means 5 or the stirring of the mixture is operated for a predetermined duration, which may be a time corresponding to 5 to 20 minutes, 5 to 15 minutes, for example about 10 minutes. The duration may be measured, for example, by a timer T, which is preferably connected to the same moving means 5. The mixture is then preferably transported by the same moving means 5 to the filtering means 6, and even more preferably, this transport is carried out by a conveyor belt.

[0126] According to a preferred variant embodiment, the filtering means 6 can be integrated into the transfer means 5, for example in the form of a filtering conveyor belt. In other words, the working surface of the conveyor belt, on which the transported mixture is placed, can be made of a filtering material. The filtering means 6 is configured to retain the PBMC-binding components and to elute the remainder of the mixture, which in fact facilitates the isolation of PBMCs bound to the above-mentioned components. For this purpose, the filtering means 6 (belonging to the group of components prone to isolating PBMCs) has, for example, a predetermined portion of an opening that prevents the passage of the PBMC-binding components and instead allows the remainder of the mixture to pass.

[0127] The selection of the cutoff of the filtration means can be easily performed by those skilled in the art depending on the size of the PBMC-binding components. In addition, the filtration cutoff should be selected to elute various blood sample cells and components that do not bind to the above-mentioned components, while retaining the component-PBMC complex. In one embodiment, when using microbeads coupled with PBMC-specific anti-antigen antibodies, the filtration means will have a cutoff selected based on the bead diameter. For example, for beads with a diameter of approximately 30 μm, the filtration means will have a cutoff that will retain the cell-bound beads and elute all those with smaller diameters. If one wishes to use reagents already available on the market, the filtration means 6 can be implemented using pluriStrainer®-type technology, which is currently available on the market.

[0128] Usefully, the isolation means 14, 5, 6, 19 may comprise a means 19 for separating the polymorphonuclear corpuscles (PBMCs) from the associated components, which is configured to favour separation between the polymorphonuclear corpuscles (PBMCs) and the components.

[0129] Advantageously, the separation means 19 may be of the filtration membrane type or of the buffer type.

[0130] The component-PBMC compound retained on the filtration means 6 is treated with a means for isolating only the PBMCs, or with a separation means 19. The isolation or separation of PBMCs from their bound components can be performed, for example, using a specific buffer (detachment buffer) that tends to favor breaking the bonds between the PBMCs and the components to which they are bound. Thus, the device can include a delivery means that is a solution suitable for detaching PBMCs from the components to which they are bound.

[0131] The PBMCs thus isolated are transported by collection means 9, comprising for example a tube or a catheter, to an analysis chamber 11. The analysis chamber 11 preferably comprises means for exposing the PBMC cytoplasm, for example a lysis buffer for the cell membranes. According to an alternative embodiment, the exposing of the cytoplasm is performed directly into the collection chamber 3.

[0132] Alternatively, to those described herein, for the purpose of obtaining PBMC separation in a blood sample, the group of components liable to isolate PBMCs may comprise a microelectromechanical system or a microfiltration parylene membrane that has a repellent effect on PBMCs and is thereby able to isolate them (Avivabio®). The membrane may be up to 36 mm 2 The membranes can have a filtering surface of about 1000 nm and a porosity of about 7% to 15% with pores of different geometric shapes (e.g., round, oval, or rectangular) with critical dimensions reduced to a few micrometers. Additionally, PBMCs can be separated in blood samples by electrolytic nickel plating techniques or by filtration through sheets of paper or plastic materials.

[0133] After isolating the PBMCs, they are processed to measure the Plin2 and / or Pnpla3 and / or Rab14 protein concentrations by a means capable of visualizing and quantifying the presence of proteins at one or more predetermined wavelengths, such as a dye-binding detection system, particularly a fluorescent dye-binding detection system. This can be achieved by a processing means 16, preferably contained in the analysis chamber 11, configured to bind the Plin2 and / or Pnpla3 and / or Rab14 proteins to the characteristic or signaling component. In one embodiment, the PBMC sample is treated with a buffer for lysis of cytoplasmic Plin2 and reacted with an anti-Plin2 monoclonal antibody, preferably coupled to a reagent that fluoresces at a predetermined wavelength, e.g., a fluorescent secondary antibody, such as Alexa Fluor® 488. The fluorescent secondary antibody is thus associated with Plin2. The device 1 further includes a third container s3 for holding the antibodies, which includes a delivery means 15 for each of the antibodies. Furthermore, the device 1 includes a spectrometer housed within the analysis chamber 11. Preferably, the spectrometer comprises a radiation source 12 and a means for acquiring images 13, both of which are housed, for example, inside the analysis chamber 11 (or collection chamber 3) and face the deposition surface of the PBMCs to be isolated and processed. The source 12 emits radiation toward the deposition surface, the radiation having a predetermined wavelength so that the fluorochrome-conjugated antibodies are visualized (or assumed to be specifically stained), thus enabling Plin2 to be selected by visual inspection. In one embodiment, the radiation source 12 is configured to emit radiation at a wavelength between 340 nm and 780 nm. Preferably, the radiation source is capable of emitting radiation at a wavelength of 488 nm. The source 12 may comprise a laser precision fluorometer or other similar devices available on the market.

[0134] Advantageously, the spectrometer 11 has the following dimensions: - a height between 15 mm and 25 mm, preferably equal to 20.1 mm, - a width between 7 mm and 18 mm, preferably equal to 12.5 mm, It may have a depth between 5 mm and 15 mm, preferably equal to 10.1 mm.

[0135] Therefore, the burden of the spectrometer 11 will be reasonable and the device 1 will be easy to carry.

[0136] Those skilled in the art will know, based on simple information in the literature, the wavelengths required to detect the fluorescent dye of interest. In addition, devices can be manufactured to emit at a variety of wavelengths, thus allowing the detection of a variety of dyes without binding the device to a particular and specific fluorescent dye.

[0137] Upon activation of the radiation source 12, the means for acquiring an image 13 is operated to acquire an image of the irradiated sample, in which the Plin2 protein and / or the Pnpla3 protein and / or the Rab14 protein can be detected and in particular quantified (qualified).

[0138] The device 1 preferably includes a control unit 7 connected to all of the above-described means and components and programmed to control and synchronize their activation in an automatic mode according to predetermined operating parameters. These operating parameters may include the duration of activation of each of the above-described means / components, as well as the blood sample pressure and / or aspiration rate, their delivery rate, the amount of anticoagulant / PBMC isolated components / fluorescent reagent / anti-Plin2 antibody delivered from each of the containers s1, s2, and s3, and / or the wavelength of the radiation source. Additionally, the control unit 7 may include or be connected to an interface means 8 configured to allow a user to control the activation of the device 1 and / or change / select the above-mentioned operating parameters. The interface means 8 may include at least one power button for the device 1.

[0139] Furthermore, the control unit 7 may comprise an image processing microprocessor programmed to identify the presence of Plin2 and / or Pnpla3 and / or Rab14 and to quantify the proteins in the images acquired by the means 13. In addition, the microprocessor may be programmed to quantify the Plin2 and / or Pnpla3 and / or Rab14 protein concentrations detected in the acquired images and output of said data. The output data may be transmitted, for example, by wired or wireless connection means contained in the device 1 itself, to an external electronic device or to display means which may for example comprise a display and be integrated into the above-mentioned interface means 8.

[0140] The microprocessor of the device 1 of the invention, in particular of the control unit 7, can further preferably be programmed to automatically compare reference data of Plin2 protein concentration obtained by analysis of the images acquired by the means 13 with reference data of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein concentration (herein referred to as reference data for the sake of simplicity), which may be Plin2 and / or Pnpla3 and / or Rab14 associated with healthy subjects, in which case the device has a dedicated diagnostic function.

[0141] The reference data mentioned above can also be stored in a memory module, for example contained in the central unit 7 and connected to a microprocessor, in which data on the Plin2 and / or Pnpla3 and / or Rab14 protein concentrations in the same patient on which the analysis is being carried out, previously detected by the device itself, are stored, in order to keep track of the progression of the disease or to evaluate the effectiveness of a therapeutic treatment.

[0142] In particular, the interface means may be configured to display a graph in which a pattern of values ​​of Plin2 and / or Pnpla3 and / or Rab14 concentration is shown and detected as a function of time.

[0143] Furthermore, the interface means 8 may be configured to automatically display a visual signal and / or reproduce an acoustic signal according to a predetermined color code based on the results of comparing the data on Plin2 protein and / or Pnpla3 and / or Rab14 concentrations obtained by the analysis with reference data.

[0144] For example, when monitoring disease progression with reference to Plin2 protein, if the Plin2 protein concentration data obtained from sample analysis is smaller than the reference data, an improvement in NAFLD is detected. This may correspond to the illumination of a green light. Alternatively, if the Plin2 protein concentration data obtained from sample analysis is greater than the reference data, a worsening of NAFLD is detected. This may correspond to the illumination of a red light and / or the sound of an audible alarm. Additionally, if the Plin2 protein concentration data obtained from sample analysis is equal to the reference data, a stagnation of the disease is detected. This may correspond to the illumination of a yellow light.

[0145] For diagnostic uses, similar systems can be used.

[0146] Furthermore, by means of the same interface means 8 it is possible to change and / or select different reference data and / or change the light settings / audio signal display settings / autoplay settings.

[0147] Preferably, the device 1 is implemented to be transported in order to enable the measurement of Plin2 and / or Pnpla3 and / or Rab14 protein concentrations also at locations allocated to a hospital or medical center. In particular, the device 1 may comprise its own power supply means, such as, for example, a rechargeable battery and / or a photovoltaic or wind charging device, making it a stand-alone device.

[0148] The conceived device has many advantages related to the size of its various components, in particular the size of the lancing means 2, the spectrometer and the pump 4. This combination of components sized in this way allows the carrying device 1 to be light and less burdensome, making it easy to transport and use.

[0149] Following what has been explained, it will be understood how the delivery device can be understood to be suitable to allow the measurement of not only Plin2 protein concentration but also other types of protein concentration in serum / plasma or PBMCs, with suitable applications and necessary modifications that are within the reach of a person skilled in the art.

[0150] In this regard, the device 1 may include a connection unit, preferably a "plug-in" unit, configured to adapt the device to other biomarkers.

[0151] According to an alternative embodiment, the device may comprise a plurality of analysis chambers 11 spaced apart therebetween, each chamber configured to measure the concentration in a blood sample of a different protein selected from among Plin2, Pnpla3 and Rab14.

[0152] This device is therefore used for various purposes, as it is possible to determine the concentration of different proteins and thereby determine the occurrence and grade of different pathologies, as explained above.

[0153] Preferably, the device may include a selection means operatively connected to the control unit 7 and configured to direct the blood sample to a correct analysis chamber 11 that is selected based on the determined protein concentration. The selection means may be controlled by the control unit 7, which is configured to send input data containing information regarding the determined protein concentration. Based on the above-mentioned input data, the selection means redirects and directs the blood sample to a particular analysis chamber 11.

[0154] Advantageously, this device may include at least one processing unit configured to execute a computer program according to one or more of the examples reported below.

[0155] Usefully, the processing unit may be operatively connected to the control unit 7. In this way, the device can use the data detected in the analysis chamber 11 to run one or more computer programs as described below.

[0156] The devices of the present invention may be implemented with suitable computer programs, for example, as described below, according to their intended use and the methods they wish to perform in the manner described and claimed herein.

[0157] Therefore, the object of the invention is the computer program as listed below.

[0158] 1. A computer program for diagnosing NAFLD and / or NASH in an individual to be analyzed, said computer program being implemented on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C0 and a third value C1 of protein concentration as defined herein, comprises the following steps: comparing the first value with the second and third values; and A computer program comprising a list of instructions for performing a step of diagnosing NAFLD when the first value is greater than or equal to the value C0 and less than the value C1, or a step of diagnosing NASH when the first value in the blood sample is greater than or equal to the value C1.

[0159] 1. A computer program for diagnosing a NAS score in an individual to be analyzed, said computer program being implemented on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C3, a third value C4 and a fourth value C5 of the concentration of Plin2 protein as defined herein, comprises the following steps: comparing the first value with the second value, the third value, and the fourth value; and NAS score 1 when the first value is equal to or greater than the cutoff value C2 and equal to or less than the cutoff value C3; NAS score 2 when the first value is greater than the value C2 and equal to or less than the cutoff value C4; A computer program comprising a list of instructions for performing a step of diagnosing an NAS score, such as an NAS score of 3, when the first value is greater than the value C4.

[0160] 1. A computer program for diagnosing the severity of NASH in an individual to be analyzed, the computer program being implemented on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of the individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C5, a third value C6 and a fourth value C7 of said Plin2 protein concentration as defined herein, comprises the following steps: comparing the first value with the second value, the third value, and the fourth value; and When the first value is greater than the value C5 and less than the value C6, the condition is mild. When the first value is greater than the value C6 and less than the value C7, the condition is moderate; A computer program comprising a list of instructions for performing a step of diagnosing the severity of NASH, such as severe when the first value is greater than the value C7.

[0161] 12. A computer program for diagnosing liver fibrosis in an individual to be analyzed, said computer program being implemented on an electronic computer, said program being provided with a first concentration value C8 of Pnpla3 protein and Rab14 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second concentration value C8 of Pnpla3 protein and Rab14 protein as defined in claim 11, said computer program comprising the following steps: comparing the first value with the second value; and A computer program comprising a list of instructions for performing a step of diagnosing liver fibrosis when the first value is greater than C8.

[0162] 1. A computer program for diagnosing the stage of liver fibrosis in an individual to be analyzed, the computer program being implemented on an electronic computer, which, when provided with a first concentration value of Pnpla3 protein and Rab14 protein in a blood sample of the individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C9, a third value C10 and a fourth value C11 of the concentrations of Pnpla3 protein and Rab14 protein, as defined herein, comprises the following steps: comparing the first value with the second value, the third value, and the fourth value; and Stage 1 when the first value is equal to or greater than the value C9 and smaller than the value C10; Stage 2 when the first value is equal to or greater than the value C10 and equal to or less than the value C11; and A computer program comprising a list of instructions for carrying out the step of diagnosing the stage of said fibrosis score, such as stage 3, when said first value is greater than said value C11.

[0163] Alternatively, the computer program described above for diagnosing NAFLD, NAS score, NASH, and severity of NASH may include one of the two instruction lists described above for diagnosing liver fibrosis and / or liver fibrosis stage.

[0164] An object of the present invention is also a computer program for monitoring the effectiveness of a therapeutic treatment of NAFLD or NASH in a patient, said program being implemented on an electronic computer and providing a first concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a moment t0, and a second concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a moment subsequent to t0, said computer program comprising the following steps: comparing the first value with the second value; and detecting the effectiveness of said therapeutic treatment if said second value is less than said first value; and a computer program for monitoring the progression of NAFLD or NASH in a patient, the computer program being implemented on an electronic computer and, when providing a first concentration value of Plin2 protein in a blood sample of the patient or in a sample of white blood cells extracted from the blood sample at a time instant t0, and a second concentration value of Plin2 protein in a blood sample of the patient or in a sample of white blood cells extracted from the blood sample at a time instant following t0, comprising the following steps: comparing the first value with the second value; and detecting an improvement in NAFLD or NASH if the second value is less than the first value; detecting a worsening of NAFLD or NASH if the second value is greater than the first value; detecting stagnation of NAFLD or NASH if the second value is approximately equal to the first value.

[0165] An object of the present invention is a device in any of the embodiments described and claimed above, which also includes at least one processing unit configured to execute a computer program according to one or more of the examples described above.

[0166] Usefully, the processing unit may be operatively connected to the control unit 7. In this way, the device can use the data detected in the analysis chamber 11 to execute one or more of the computer programs described above.

[0167] An object of the invention is also the use of a device in any one of the embodiments described above for carrying out the method that is the object of the invention.

[0168] Finally, an object of the present invention is a therapeutic method for the treatment of patients suffering from NAFLD, in which the monitoring described herein is carried out.

[0169] example method Nineteen obese subjects (10 women and 9 men) with NAFLD had a mean age of 38.5 ± 1.9 years and a BMI of 36.79 ± 4.43 kg / m 2 ) were enrolled and underwent weight loss surgery. At enrollment, peripheral venous blood samples containing EDTA were collected after a 12-hour overnight fast. Patients signed informed consent for the study and surgical intervention.

[0170] A medical history was collected for all subjects. Objective examinations including height, weight, and anthropometry were also performed. Weight (kg) was proportional to height (m 2 ) to calculate body mass index (BMI). A detailed list of medications used was compiled.

[0171] The exclusion list included: (1) regular and / or excessive alcohol intake (more than 20 g of alcohol per day for women and more than 30 g of alcohol per day for men), (2) clinical evidence of NAFLD secondary to iatrogenic gastrointestinal disease or immunodeficiency disease (HIV infection), (3) non-NAFLD liver disease, including hepatitis B or C, or hemochromatosis, (4) Wilson's disease, (5) glycogen storage disease, (6) alpha-1 antitrypsin deficiency, (7) autoimmune hepatitis, (8) cholestatic liver disease, (9) associated cardiovascular, gastrointestinal, or respiratory disease, or any hormonal disorder, (10) clinical evidence of decompensated liver disease (Child-Pugh score greater than 7), (11) current drug abuse state, (12) associated systemic disease, and (13) pregnancy.

[0172] Liver biopsies were obtained during surgery.

[0173] All subjects underwent an oral glucose tolerance test (OGTT) to measure blood insulin and glucose levels (blood glucose) at 0, 30, 60, 90, 120, 150, and 180 minutes. Insulin sensitivity was measured as OGIS (acronym for oral glucose insulin sensitivity), a method that allows for the calculation of insulin sensitivity from the OGTT. OGIS yields an insulin sensitivity index similar to that obtained using the clamp method (Mari A, Pacini G, Brazzale AR, Ahren R. Comparative evaluation of simple insulin sensitivity methods based on the oral glucose tolerance test. Diabetologia 2005;48:748-751).

[0174] Liver histology Formalin-preserved liver biopsies were thoroughly washed with 60% isopropanol, which was then allowed to evaporate. Oil Red O (ORO) solution was added for 10 minutes, then removed, and the samples were washed four times with water. Upon removal of the ORO solution, the sections were incubated in 100% isopropanol. Monocytes were also subjected to the same ORO staining.

[0175] The samples were then observed under an LSM510 confocal microscope equipped with appropriate filters.

[0176] Furthermore, to assess the rate of steatosis, biopsies obtained during surgery were fixed in 10% formalin, contained in paraffin blocks, and prepared from sections stained with hematoxylin and eosin and mounted on archival slides. Slide reading was performed by a blinded hepatic pathologist.

[0177] 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) was used to assess histological scoring and NAFLD / NASH (nonalcoholic steatohepatitis) stage. Specifically, steatosis was defined as 0, absent (<1%); 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, greater than 75% intralobular fat. Inflammation was scored as 1, mild (lymphocytes isolated (scattered) or aggregated into small formations within portal ducts and lobules); 2, moderate (similar to grade 1, but no significant portal or lobular infiltrates); and 3, severe (similar to grade 2, but no significant inflammation yet). Fibrosis was staged as 0 for absent, 1 for centrilobular pericellular, 2 for periportal and pericellular, 3 for bridging fibrosis, and 4 for cirrhosis.

[0178] Isolation of monocytes PBMCs were obtained from whole blood by standard gradient centrifugation on Ficoll-Hypaque (GE Healthcare Bio-Sciences, Piscataway, NJ), and then washed and monocytes were isolated using a Pan Monocyte Isolation Kit.

[0179] Hepatocyte isolation The liver biopsy fragments were minced and washed in HBSS to remove traces of blood. They were then transferred to a 50 mL test tube containing EGTA buffer (HBSS, 0.5 mM EGTA, 0.5% BSA) and agitated 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 agitated 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. The cell suspension was centrifuged at 80 rpm for 5 minutes at 4°C, and the supernatant was discarded.

[0180] Lipid droplet staining Isolated monocytes and hepatocytes were incubated with 4% formalin for 20 minutes and stained with Nile red (100 ng / mL). Nuclei were stained with DAPI.

[0181] Photographs were taken with a spinning disk confocal microscope such as a Crest X-Light Confocal Imager (Germany), and images were analyzed with MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices).

[0182] Plin2 measurement Monocytes and liver biopsies were homogenized in RIPA buffer containing a protease inhibitor cocktail. The homogenates were centrifuged at 13,000 rpm for 30 minutes at 4°C. Protein content was measured using a 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 antibody (LS-BIO, Seattle, WA) and anti-β-actin. Qualitative and quantitative analysis was performed using a Chemidoc XRS Image system and Image Lab 5.0 software (Bio-Rad Laboratories, Hercules, CA). All data were normalized to β-actin levels (8H10D10).

[0183] statistics Data are expressed as mean ± SD unless otherwise specified. Spearman's correlation was used to measure the degree of correlation between two variables. Bland-Altman plots were used to measure the agreement between the two methods. Bland-Altman results show the difference and mean values ​​obtained with the two methods (Plin2 levels in monocytes and liver). The standard deviation (SD) of the difference between the two methods is the SD bias. The tolerance was measured as the mean bias, i.e., the mean difference between the two measurement methods ± 1.96 of the SD bias.

[0184] The sensitivity and specificity of the method were studied by neural network analysis. The parameters of the neural network analysis are reported below: two input layers are used: Plin2 in monocytes and age in controls. Only one hidden layer containing 6 units and a hyperbolic (tangent) activation function. The output layer is the dependent variable, i.e., NAS level. Activation function: Softmax, error function: cross entropy.

[0185] 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-specificity) on the Y-axis. The AUC closest to 1 is the best predictive value of the test. Statistical analysis was performed using SPSS version 13.

[0186] result The grade of fatty liver was assessed by liver biopsy and by the NAFLD Activity Score (NAS), defined above, a histological method for defining the grade of NAFLD; the mean steatosis grade of all patients studied was 2.42 ± 1.17, with subjects presenting with a steatosis grade of 1 to 4.

[0187] To demonstrate that ectopic lipid accumulation also occurs in monocytes, Nile red was used to stain liver biopsies and monocytes (Figure 1, panel A), in which lipids aggregate into larger droplets.

[0188] Plin2 expression in monocytes strongly and positively correlates with Plin2 expression in the liver (R=0.84, P<0.0001). An example of Plin2 protein expression by Western blot is reported in Figure 1, panel B.

[0189] FIG. 2 instead reports ORO staining in liver sections and monocytes of the same subjects.

[0190] Plin2 levels in the liver correlated well with the grade of fatty liver (R=0.91, P<0.001), and Plin2 levels in monocytes correlated well with the grade of fatty liver (R=0.89, P<0.001).

[0191] Figure 3 reports a Bland-Altman graph comparing Plin2 levels in monocytes and liver after two administration methods. The difference between the values ​​obtained in the two measurements is reported on the Y-axis, while the mean is on the Y-axis. The dotted line reports the 95% agreement boundary between the two measurements. The graph demonstrates that all points lie within the agreement boundary, which means that the method of the present invention is valid.

[0192] Alternatively, we found a good correlation between Plin2 levels measured in monocytes by cytofluorometry, expressed as mean fluorescence intensity (MFI, Figure 4), and by Western blot (R = 0.92, P < 0.0001).

[0193] Plin2 levels (MFI) in PBMCs, which were 48.88±5.80 (SEM), demonstrate an excellent correlation (R=0.98, P=0.0004) with levels in monocytes, which were 42.90±5.77 (SEM).

[0194] As evidence of a correlation between the severity of nonalcoholic fatty liver disease and Plin2 levels, Plin2 levels in both liver and monocytes were found to be highly inversely correlated with insulin sensitivity, expressed as OGIS, and the regression equation for monocytes was: OGIS=44.59 * Plin2+411.3, R of 0.91 2 and with P<0.0001.

[0195] OGIS is an acronym for Oral Glucose Insulin Sensitivity Index, and its unit of measurement is mL x min -1 ×m -2 Plin2 is measured by Western blot in units relative to the β-actin reference protein.

[0196] Patient analysis Ninety-one subjects suspected of having NASH underwent liver biopsy, while 21 subjects with a normal body mass index underwent elective cholecystectomy and those with a negative liver ultrasound test during surgery (controls) underwent liver core biopsy. The age range was 18-67 years, with 55% being female and 45% being male.

[0197] Histological examination highlighted different stages of NAFLD activity score (NAS).

[0198] Plin2 levels in circulating monocytes analyzed by cytofluorometry were used to predict NAS.

[0199] One of the controls had NAS=0 and the other had NAS=1.

[0200] Cytofluorescence analysis Monoclonal antibody against Plin2 was obtained from LS-BIO (Seattle, WA), fluorophore-conjugated secondary monoclonal antibody Alexa Fluor 488 was obtained from Life Technology (Carlsbad, CA), and anti-CD14-ECD antibody was obtained from Beckman Coulter (Brea, CA).

[0201] Monocytes were identified using anti-CD14-ECD antibody, which was used to identify the monocyte population among all polymorphonuclear cells. Monocytes were fixed and permeabilized by standard techniques, as reported below.

[0202] One part solid / permeabilization concentrate was mixed with three parts fixation / permeabilization diluent. The cell pellet (2 x 10 cells) was resuspended in 300 μL of 1x permeabilization buffer and incubated for 45 minutes at 4°C. It was washed with 1x phosphate-buffered saline (PBS), centrifuged, and the cells were resuspended in 100 μL of 1x PBS. The cells in suspension were divided into two test tubes (50 μL each). Tube 1: Cells in suspension were stained with IgG antibody Alexa Fluor 488 (1:2000) for 20 minutes at room temperature in the dark. This tube was the negative control. Tube 2: Cells in suspension were stained with anti-Plin2 (1 μl in 50 μl) and then with IgG antibody Alexa Fluor 488 and anti-CD14-ECD antibody (4 μl in 50 μl) for 20 minutes at room temperature in the dark.

[0203] They were washed, centrifuged, and resuspended in 500 μL of 1×PBS before proceeding to cytofluorimetric analysis.

[0204] Therefore, monocytes were stained for Plin2 using a monoclonal antibody against Plin2 and a fluorophore-conjugated secondary antibody (Alexa Fluor 488) bound to the anti-Plin2 antibody.

[0205] The instrument used for cytofluorometry was an FC 500 (Beckman Coulter, Brea, CA), and data were analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0206] Plin2 detection is obtained by a fluorescent tracer that generates a signal that is acquired by the photodiode of a cytofluorometer and converted in terms of mean fluorescence intensity (MFI).

[0207] MFI is proportional to the number of antibodies that recognize and bind to a cellular antigen (in this case, Plin2), allowing for protein quantification (Mizrahi O., Shalom EI, Baniyash M., Klieger Y. Quantitative flow cytometry: concerns and recommendations in clinic and research. Cytometry B Clin Cytom. 2017).

[0208] Histologically, NAFLD is defined when steatosis affects more than 5% of hepatocytes, whereas NASH is defined instead by the presence of steatosis plus any grade of hepatocyte swelling and degeneration, regardless of number, and lobular inflammatory infiltration. The NAFLD Activity Score (NAS) results from the combination of steatosis, hepatocyte swelling and degeneration, and lobular inflammatory infiltration of liver tissue. From the table below, it can be seen that the sum of the individual components achieves a maximum score of 8.

[0209] [Table 6] (Kleiner DE, Brunt EM, Van Natta M., Behlinh C., Contos MJ, Cummings OW, Ferrell LD, Liu Y.-C., Torbenson MS, Unalp-Arida A., Yeh M., McCullough AJ, Sanyal AJ for the Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology 41:1313-1321,2005.)

[0210] Plin2 (mean fluorescence intensity [MFI]) levels at different NAS stages are reported in the following table: [Table 7]

[0211] As reported below, the correlation between mean Plin2 levels (MFI) and NAS stage is excellent.

[0212] The independent variable on the x-axis is the mean value of Plin2, while the dependent variable is the NAS stage (Fig. 7).

[0213] [Table 8]

[0214] The R2 was very high (0.985) with a significance of P<0.0001, indicating a good model for predicting NASH histological stage, i.e., NAS.

[0215] [Table 9]

[0216] The ability of Plin2 to predict different NAS stages was studied by neural network analysis.

[0217] The neural network analysis parameters are reported below: Two input layers were used: Plin2 in monocytes and subject age. Only one hidden layer containing 6 units and a hyperbolic (tangent) activation function. The output layer is the dependent variable, i.e., NAS level. Activation function: Softmax, error function: cross entropy.

[0218] [Table 10]

[0219] This design predicted incorrect NAS values ​​only 6.3% of the time during training and 11.35% of the time during the 0.05 s test, as reported below.

[0220] [Table 11]

[0221] The data demonstrate that the area under the curve (AUC) of the receiver operating characteristic curve (ROC) is very high, ranging from a minimum of 0.974 to a maximum of 1. All stages are predictive in terms of excellent sensitivity and specificity.

[0222] [Table 12]

[0223] The importance of the Plin2 variable in the model is reported below as 68.3%, which reaches 100% after normalization, while the importance of the second variable used in the model, namely age, is 31.7%, which reaches 46.3% after normalization.

[0224] [Table 13] (See Figure 8)

[0225] Repeated inference, i.e., multiple comparison analysis adjusted for multiple comparisons, demonstrates highly significant differences in the various NAS stages, see table below.

[0226] [Table 14]

[0227] [Table 15-1] [Table 15-2]

[0228] Taken together, Plin2 in monocytes is highly predictive of NASH severity stage (i.e., NAS score), allowing invasive tests such as biopsy to be replaced by tests in peripheral blood.

[0229] fibrosis Patatin-like phospholipase domain-containing protein 3 (Pnpla3) (Sigma Aldrich SAB1401851) and the Ras-related protein Rab-14 (Sigma Aldrich R0656) were administered to a total of 132 subjects, always with cytofluorimetric analysis in monocytes and histological examination of liver and monocyte isolation.

[0230] Below, the mean values ​​(MFI) for each stage of SAF fibrosis range from F0 to F4 (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).

[0231] [Table 16]

[0232] [Table 17]

[0233] Next, neural network analysis was performed to calculate the area under the receiver operating characteristic curve (AUC) using Pnpla3 and Rab14 monocytes 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 exaggerated. The model output yielded the fibrosis grade (SAF F), the activation function was softmax, and the error function was cross-entropy.

[0234] [Table 18]

[0235] [Table 19]

[0236] [Table 20]

[0237] The error rate during training of the model was 14.3%, the error rate during testing was 0%, and the time used was 0.05 minutes.

[0238] The area under the curve (AUC) of the ROC analysis is reported in the following table: [Table 21]

[0239] Thus, stage 0, i.e., the absence of fibrosis, is predicted in 100%, stage 1 in 95%, stage 2 in 100%, and stage 3 in 95.5%.

[0240] The importance of the independent variables in the model (Pnpla3 monocytes and Rab14 monocytes) is 62%, but when normalized it becomes 100%; the importance of Pnpla3 monocytes is 29.7%, but when normalized it reaches 47.9% for Rab14 monocytes; the importance of the presence or absence of diabetes is 0.84%, but when normalized it becomes 13.5%.

[0241] [Table 22] (See Figure 9)

[0242] Therefore, the use of Pnpla3 and Rab14 makes it possible to obtain very accurate information regarding fibrosis.

[0243] However, Plin2 alone also demonstrates fibrosis well.

[0244] [Table 23]

[0245] [Table 24]

[0246] [Table 25]

[0247] Percentage of incorrect predictions during training: 14.3%, during testing: 20%.

[0248] In defining fibrosis, the AUCs of the ROC reported below for Plin2 are 98.9% for stage 0, 94% for stage 1, 97% for stage 2, 98.8% for stage 3, and 100% for stage 4.

[0249] [Table 26]

[0250] As reported in the table and Figure 10, the significance of Plin2 in monocytes at the time of fibrosis diagnosis is 49.2%, which is normalized to 96.8%. The significance of the presence or absence of diabetes is 50.8%, which is normalized to 100%.

[0251] [Table 27]

[0252] [Table 28]

[0253] [Table 29-1] [Table 29-2]

Claims

1. 1. A method for the diagnosis of non-alcoholic steatohepatitis (NASH), comprising: a. measuring the concentration of Plin2 protein in a blood sample of an individual or in a sample of white blood cells extracted from said blood sample; b. comparing the value obtained at time point a with the concentration value of Plin2 protein in a blood sample of a healthy individual or in a sample of white blood cells extracted from said blood sample; c) A method comprising a step of providing a diagnostic indicator in which NASH is diagnosed when the value measured in a is greater than the value measured in b.

2. 1. A method for the diagnosis of NASH, comprising: a. Measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring the concentration values ​​of Plin2 protein in a group of blood samples from patients with NASH and healthy patients, and identifying a cut-off value C1 of said concentration between patients with NASH and healthy patients; c. comparing the value obtained at time point a with the cut-off value obtained at time point b; d. A method comprising a step of providing a diagnostic indicator in which NASH is diagnosed when the value obtained in a is equal to or greater than the value C1.

3. 1. A method for diagnosing the NAS score of a patient suffering from NAFLD or NASH, comprising: a. Measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NAFLD or NASH and healthy patients, wherein in the population including samples from patients with NAFLD, NAS score values ​​are histologically defined in the patients as NAS1, NAS2, and NAS3 based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; A cut-off value C2 of the concentration between healthy patients and patients with NAFLD having a NAS score of 1; a cutoff value C3 of the concentration between patients suffering from NAFLD with a NAS score of 1 and patients suffering from NAFLD with a NAS score of 2; and Identifying a cut-off value C4 of the concentration between patients suffering from NAFLD with a NAS score of 2 and patients suffering from NASH with a NAS score of 3; c. comparing the value obtained at time point a with the cut-off value obtained at time point b; d. The NAS score is calculated by: When the value obtained in a is equal to or greater than the cutoff value C2 and equal to or less than the cutoff value C3, the NAS score is 1; When the value obtained in a is greater than the value C2 and equal to or less than the cut-off value C4, the NAS score is 2; When the value obtained in a is greater than the value C4, the patient is diagnosed with an NAS score of 3, which is a diagnostic indicator.

4. 1. A method for diagnosing the severity of NASH, comprising: a. Measuring the concentration of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample; b. Measuring Plin2 protein concentration values ​​in a population of blood samples from patients with NASH and healthy patients, wherein the severity of the condition in said population, including samples from patients with NASH, is histologically defined in said patients as mild, moderate, or severe based on the percentage of steatosis in liver cells, the occurrence of swollen hepatocytes, the presence of lobular inflammation, and the presence of portal vein inflammation; a cutoff value C5 of the concentration between healthy patients and patients suffering from mild NASH; A cutoff value C6 of the concentration between patients with mild NASH and patients with moderate NASH; and Identifying a cut-off value C7 of said concentration between patients suffering from moderate NASH and patients suffering from severe NASH; c. comparing the value obtained at time point a with the cut-off value obtained at time point b; d. The severity of NASH is assessed by: When the value obtained in a is greater than the value C5 and less than the value C6, the condition is mild. When the value obtained in a is greater than the value C6 and less than the value C7, the condition is moderate; When the value obtained in step a is greater than the value C7, the condition is diagnosed as severe, providing a diagnostic indicator.

5. The method according to any one of claims 1 to 4, wherein said concentration value of Plin2 protein is measured by Western blot, or cytofluorimetric analysis, or ELISA, or quantitative PCR.

6. 1. A method for the diagnosis of NASH, comprising: a. Measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and binding specifically to Plin2 and not to other proteins; b. Comparing the value obtained at time point a with a concentration cut-off value expressed in terms of mean fluorescence intensity (MFI) equal to 2.7 MFI and a concentration cut-off value equal to 1.0 MFI; d. A method comprising a step of providing a diagnostic indicator in which NASH is diagnosed when the value obtained in a is greater than 2.7 MFI.

7. 1. A method for diagnosing a NAS score in a patient suffering from NASH, comprising: a. Measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and binding specifically to Plin2 and not to other proteins; c. Comparing the value obtained at time point a with cut-off values ​​of said concentrations, expressed in terms of mean fluorescence intensity (MFI), equal to 1.0 MFI, 1.4 MFI and 2.7 MFI; d. A method comprising the steps of: indicating a diagnostic indicator that diagnoses the patient with an NAS score of 1 when the value obtained in step a is equal to or greater than the cutoff value of 1.0 MFI and equal to or less than the cutoff value of 1.4 MFI; indicating a diagnostic indicator that diagnoses the patient with an NAS score of 2 when the value obtained in step a is greater than the cutoff value of 1.4 MFI; or indicating a diagnostic indicator that diagnoses the patient with an NAS score of 3 when the value obtained in step a is greater than the cutoff value of 2.7 MFI.

8. 1. A method for diagnosing the severity of NASH, comprising: a. Measuring the concentration value of Plin2 protein in a blood sample or in a sample of white blood cells extracted from said blood sample, said value being measured by cytofluorometric analysis using a monoclonal antibody labeled with a suitable fluorescent dye and binding specifically to Plin2 and not to other proteins; c. Comparing the value obtained at time point a with cut-off values ​​of said concentrations, expressed in terms of mean fluorescence intensity (MFI), equal to 2.7 MFI, 4 MFI and 6.3 MFI; d. A method comprising a step of providing a diagnostic indicator for diagnosing mild NASH when the value obtained in a is greater than the cutoff value of 2.7 MFI and equal to or less than the cutoff value of 4 MFI, moderate NASH when the value obtained in a is greater than the cutoff value of 4 MFI and equal to or less than the cutoff value of 6.3 MFI, and severe NASH when the value obtained in a is greater than 6.3 MFI.

9. e. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample or a sample of white blood cells extracted from the blood sample at time point a to diagnose NASH; f. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a group of blood samples from patients with liver fibrosis and healthy patients, and identifying a cutoff value C8 of the concentration values ​​between patients with liver fibrosis and healthy patients; g. Comparing the value obtained at time point e with the cut-off value obtained at time point f; h. The method further comprises the step of providing a diagnostic indicator, wherein the value obtained at time point e is greater than the C8 value, and the diagnosis is made as liver fibrosis; The method according to any one of claims 2 to 8.

10. e. Measuring the protein concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample at time point a or in a sample of white blood cells extracted from said blood sample to diagnose NASH; f. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a population of blood samples from healthy patients and in a population of blood samples from patients suffering from liver fibrosis, wherein the severity of the condition is histologically defined as stage 1, stage 2, or stage 3 based on the location and extent of liver fibrosis; the cut-off value C9 of the concentration between healthy patients and patients with stage 1 liver fibrosis; a cutoff value C10 of the concentration between patients with stage 1 liver fibrosis and patients with stage 2 liver fibrosis; and Identifying a cut-off value C11 of said concentration between patients suffering from stage 2 liver fibrosis and patients suffering from stage 3 liver fibrosis; g. Comparing the value obtained at time point e with the cut-off value obtained at time point f; h. Stage 1 when the value obtained at time e is equal to or greater than the value C9 and less than the value C10; Stage 2 when the value obtained at time e is equal to or greater than the value C10 and equal to or less than the value C11; Stage 3 when the value obtained at time e is greater than the value C11; The method further comprises the step of diagnosing the stage of liver fibrosis as a diagnostic indicator, The method according to any one of claims 2 to 9.

11. The concentration values ​​of Pnpla3 protein and Rab14 protein are measured by Western blot, or cytofluorimetric analysis, or ELISA, or quantitative PCR; or The method of claim 9 or 10, wherein the concentration values ​​of Pnpla3 protein and Rab14 protein are measured by cytofluorometry using a monoclonal antibody that specifically binds to Pnpla3 but not to other proteins, and a monoclonal antibody that specifically binds to Rab14 but not to other proteins, labeled with a suitable fluorescent dye.

12. e. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample or a sample of white blood cells extracted from said blood sample at time point a for diagnosing NASH, said values ​​being measured by cytofluorometry using a monoclonal antibody that specifically binds to Pnpla3 but not to other proteins, and a monoclonal antibody that specifically binds to Rab14 but not to other proteins, both labeled with suitable fluorescent dyes; g. Comparing the value obtained at time point e with a cut-off value, expressed in terms of mean fluorescence intensity (MFI), of 1.24 MFI or greater; h. The method further comprises providing a diagnostic indicator, wherein a value obtained at time point e of 1.24 MFI or greater diagnoses liver fibrosis; The method according to any one of claims 2 to 11.

13. e. Measuring the concentration values ​​of Pnpla3 protein and Rab14 protein in a blood sample or a sample of white blood cells extracted from said blood sample at time point a for diagnosing NASH, said values ​​being measured by cytofluorometry using a monoclonal antibody that specifically binds to PNPLA but not to other proteins, and a monoclonal antibody that specifically binds to Rab14 but not to other proteins, both labeled with suitable fluorescent dyes; g. Comparing the value obtained at time point a with cut-off values ​​for said concentrations, expressed in terms of mean fluorescence intensity (MFI), equal to 1.24 MFI, 2.3 MFI, and 3.10 MFI; h. providing a diagnostic indicator, in which a value obtained at time point e of 1.24 MFI or more and less than 2.4 MFI indicates mild stage 1 liver fibrosis, a value obtained at time point e of 2.4 MFI or more and less than 3.10 MFI indicates stage 2 liver fibrosis, and a value obtained at time point e of 3.10 MFI or more indicates stage 3 liver fibrosis; The method of any one of claims 2 to 11, further comprising:

14. 1. A method for monitoring the effectiveness of a therapeutic treatment of NASH in a patient, comprising: a. measuring a concentration value of Plin2 protein in a blood sample of an individual suffering from NASH or in a sample of white blood cells extracted from said blood sample at the time t0 when said monitoring is carried out; b. measuring concentration values ​​of Plin2 protein in a blood sample of an individual suffering from NASH or in a sample of white blood cells extracted from said blood sample at one or more instants tn, where n is an integer greater than 0 and each tn corresponds to an instant after t; a decrease in the concentration of Plin2 protein at one or more of said moments tn indicates the effectiveness of said therapeutic treatment.

15. 1. A method for monitoring the progression of NASH in a patient, comprising: a. measuring a concentration value of Plin2 protein in a blood sample of an individual suffering from NASH or in a sample of white blood cells extracted from said blood sample at the time t0 when said monitoring is carried out; b. A method comprising the step of measuring concentration values ​​of Plin2 protein in a blood sample of an individual suffering from NASH or in a sample of white blood cells extracted from said blood sample at one or more instants tn, where n is an integer greater than 0 and each tn corresponds to an instant after t0, and a decrease in the concentration of Plin2 protein at one or more of said instants tn is indicative of an improvement in NASH, whereas an increase in the concentration of Plin2 protein at one or more instants tn is indicative of a worsening of NASH.

16. The concentration value of Plin2 protein is measured by Western blot, or cytofluorimetric analysis, or ELISA, or quantitative PCR; or 16. The method according to claim 14 or 15, wherein the concentration value of Plin2 protein is measured by cytofluorometry using a monoclonal antibody labeled with a suitable fluorescent dye and which specifically binds to Plin2 and not to other proteins.

17. the blood sample is a peripheral blood sample, and / or The method according to any one of claims 1 to 16, wherein the white blood cells are polymorphonuclear cells and / or monocyte cells.

18. 1. A computer program for diagnosing NASH in an individual to be analyzed, said computer program being implemented on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second concentration value of Plin2 protein in a blood sample of a healthy individual or in a sample of white blood cells extracted from said blood sample, comprises the following steps: comparing the first value with the second value; and A computer program comprising a list of instructions for performing the step of diagnosing NASH when the first value is greater than the second value.

19. 1. A computer program for diagnosing NASH in an individual to be analyzed, said computer program being implemented on an electronic computer, said computer being provided with a first concentration value of Plin2 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C0 and a third value C1 of protein concentrations as defined in claim 2, said computer program comprising the following steps: comparing the first value with the second and third values; and A computer program comprising a list of instructions for performing a step of diagnosing NASH when the first value in the blood sample is equal to or greater than the value C1.

20. 1. A computer program for diagnosing the severity of NASH in an individual to be analyzed, said computer program being executed on an electronic computer, said computer being provided with a first concentration value of Plin2 protein in a blood sample of said individual to be analyzed or in a sample of white blood cells extracted from said blood sample, and a second value C5, a third value C6 and a fourth value C7 of the concentration of Plin2 protein as defined in claim 4, said computer program comprising the following steps: comparing the first value with the second value, the third value, and the fourth value; and mild when the first value is greater than the value C5 and less than the value C6; moderate when the first value is greater than the value C6 and less than the value C7; diagnosing the severity of NASH as severe when the first value is greater than the value C7.

21. 1. A computer program for monitoring the effectiveness of a therapeutic treatment of NASH in a patient, the computer program being implemented on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a time instant t0, and a second concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a time instant subsequent to t0, comprises the following steps: comparing the first value with the second value; and detecting the effectiveness of the therapeutic treatment if the second value is less than the first value.

22. 1. A computer program for monitoring the progression of NASH in a patient, the computer program being executed on an electronic computer, which, when provided with a first concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a time instant t0, and a second concentration value of Plin2 protein in a blood sample of said patient or in a sample of white blood cells extracted from said blood sample at a time instant subsequent to t0, comprises the following steps: comparing the first value with the second value; and detecting an improvement in NASH if the second value is less than the first value; detecting a worsening of NASH if the second value is greater than the first value; detecting NASH stagnation if the second value is approximately equal to the first value.

23. A device (10) for the automated measurement of Plin2 and / or Pnpla3 and / or Rab14 protein concentrations in a patient's blood sample, comprising: - puncturing means (2) facilitating piercing the skin of the individual from whom said blood sample is to be taken; - means (14, 5, 6, 19) for isolating polymorphonuclear leukocytes (PBMCs) in a blood sample, comprising at least means (19) for separating said polymorphonuclear leukocytes (PBMCs) from associated components, and configured to favor a separation between said polymorphonuclear leukocytes (PBMCs) and said components, said means (19) being of the membrane filtration type or of the buffer solution type; at least one spectrometer (12, 13) comprising at least one analysis chamber (11) and means (16, 12, 13) for measuring the concentration of Plin2 and / or Pnpla3 and / or Rab14 proteins in the cytoplasm of said polymorphonuclear leukocytes (PBMC), said means (16, 12, 13) comprising: - means (16) for treating the polymorphonuclear cells (PBMCs) isolated by the isolation means (14, 5, 6, 19), which are configured to allow binding of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein, when present, with a specific reagent for said protein, said reagent being a fluorescent dye or having a specific color emission at one or more predetermined wavelengths, and at least one spectrometer (12, 13), a radiation source (12) configured to emit radiation at a predetermined wavelength at the isolated and treated polymorphonuclear leukocytes (PBMCs) so that the reagent emits a fluorescent dye or exhibits the specific color; means (13) for acquiring an image of a sample illuminated by a radiation source (12); At least one spectrometer (12, 13) comprising At least one spectrometer including: a control unit (7) connected to said isolation means (14, 5, 6, 19) and to said measurement means (16, 12, 13), programmed to control and synchronize the actuation of said isolating means (14, 5, 6, 19) and said measuring means (16, 12, 13) in an automatic mode according to predetermined operating parameters; a control unit (7) for automatically comparing the concentration data of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein measured in the cytoplasm of the polymorphonuclear leukocytes (PBMC) with at least one reference data of Plin2 protein and / or Pnpla3 protein and / or Rab14 protein concentration, A device, said device further comprising at least one processing unit configured to execute one or more of the computer programs of claims 18 to 22.

Images