RU2697202C1 - Method for differential diagnosis of fatty liver disease of alcoholic and non-alcoholic genesis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for differential diagnosis of fatty liver disease of alcoholic and non-alcoholic genesis. That is ensured by exposing a patient's erythrocyte suspension to an inhomogeneous alternating electric field. It is followed by video recording of the change in the size of each erythrocyte in the period of exposure to the electric field and after its disconnection. Computer analysis of the obtained video frames is used to program the value of the average deformation amplitude in the erythrocyte sample (M1). Ethanol solution is then added to said suspension. That is followed by video recording of a change in the size of each red cell treated with ethanol in the period of exposure to the electric field and after its disconnection. Based on the computer analysis, the average deformation amplitude in the erythrocyte sample is calculated after exposure in the ethanol solution (M2). Difference M1 - M2 is determined. If observing a positive and statistically significant difference M1 and M2, a non-alcoholic fatty liver disease is stated. If there is a negative and significant difference M1 and M2, the presence of alcoholic fatty liver disease is stated.

EFFECT: invention enables differential diagnosis of fatty liver disease.

1 cl, 2 dwg, 1 tbl, 2 ex

Description

The invention relates to medicine and can be used in medicine, namely: hepatology and gastroenterology as a method for the differential diagnosis of fatty liver disease of alcoholic and nonalcoholic origin.

An important criterion for distinguishing non-alcoholic fatty liver disease (NAFLD) from alcoholic fatty liver disease (ALFL) is the use by patients of alcohol in hepatotoxic doses. In most patients, NAFLD is associated with metabolic syndrome (MS) [5]. In the pathogenesis of alcoholic (AFLD) and nonalcoholic (NAFLD) fatty liver disease that arose against the background of the metabolic syndrome, a coincidence of most of the metabolic pathways is observed, which creates difficulties in diagnosis [4, 6]. In addition, changes in the elasticity, viscoelastic characteristics (VUH) of erythrocytes, and their ability to deform are noted [1, 2].

The prior art. In the diagnosis of fatty disease, a number of methods are widely used:

- liver biopsy;

- ultrasound procedure;

- laboratory parameters of red blood cells;

- biochemical tests.

Liver biopsy, being the "gold standard" for determining the degree of fibrosis and cirrhosis, is unacceptable for the differential diagnosis of fatty liver disease due to a number of disadvantages, the main of which are invasiveness, biopsy errors, the influence of the "human factor" (different interpretation of the results of one biopsy sample) , the dependence of the result on the quality of the biopsy, as well as the error inherent in the biopsy procedure itself (fragmentation due to varying degrees of changes in different parts of the liver tissue neither). It is often impossible to recommend a biopsy for clarification or final diagnosis because of the complexity of the procedure, the need for hospitalization, the high risk of complications, and the high cost of the procedure. In addition, the morphological picture with alcoholic and non-alcoholic fatty liver damage is similar, Mallory bodies, which were previously considered specific for alcoholic hepatitis, are also found in non-alcoholic steatohepatitis. Due to the invasiveness, the large error of the histological examination associated with the “errors of getting” the needle during puncture biopsy of the liver, the difference in the interpretation of the results, more accurate and reliable diagnostic methods are currently being paid more attention to the early diagnosis of pathological processes [3, 4].

Ultrasound examination (ultrasound) of the liver in addition to the size, structure, nature of the parenchyma, using special equipment, allows you to determine the degree of fatty infiltration and fibrosis. However, ultrasound does not allow to differentiate alcoholic and nonalcoholic fatty liver disease. In addition, the interpretation of ultrasound data in practice is difficult due to obtaining difficult to reproduce data with large differences in interpretation. The FibroScan ultrasound elastometry method also allows you to evaluate the Controlled Attenuation Parameter (CAP ™), a controlled attenuation parameter that is used for non-invasive assessment and quantification of steatosis. ATS ™ is the amount of attenuation of ultrasound, which corresponds to a decrease in the amplitude of ultrasonic waves as they propagate through the liver tissue. Using this parameter expands the range of non-invasive methods of examination and subsequent monitoring of patients with liver diseases, however, alcohol and non-alcoholic liver damage cannot be discriminated against.

Laboratory indicators of red blood cells in the diagnosis of chronic alcohol consumption are: an increase in the average corpuscular volume, an increased level of triangulocytes, the appearance of an abnormal phospholipid - phosphatidylethanol in the membranes of red blood cells, but the change in these parameters is not specific for fatty liver disease of alcoholic origin. [7-9].

Biochemical tests for the diagnosis of alcoholic and nonalcoholic liver damage are based on methods that differ from each other depending on the etiology of the process:

- a diagnostic method based on the presence of concomitant biochemical disorders, in particular, lipid, carbohydrate metabolism. These include FibroTest, FibroMaxTest, Fibrometer. So, as part of FibroMax there are a number of non-invasive tests to detect the presence of steatohepatitis (SteatoTest), non-alcoholic (NeshTest) and alcoholic (AshTest) forms of steatohepatitis [10];

- The SteatoScreen method, which allows confirming or refuting liver steatosis, established by ultrasound, as well as identifying signs of liver steatosis in high-risk individuals. For this method, the correlation with histological signs of liver steatosis was studied, which allows using it with a high degree of reliability and non-invasively determining the presence of liver steatosis in patients. [11, 12].

The disadvantages of all of the above methods are the high complexity and cost, low availability of equipment, the need for special training of personnel implementing the methods, and the need to use a wide range of different reagents. These factors determine the impossibility of using these methods for mass diagnostics, for screening purposes. The diagnostic accuracy of the disease often depends on the severity of hemolysis, cytolysis, cholestasis syndromes, the presence of a concomitant infection in the patient, which may affect the level of the studied biochemical parameters and complicate the interpretation of the result, especially in light of the differential diagnosis of fatty liver disease of alcoholic and non-alcoholic origin. The above methods do not provide for measuring the amplitude of erythrocyte deformation, their viscoelastic characteristics, after adding alcohol to the cell suspension and exposing the cells to it before measurements.

The closest analogues (prototype) is a method for determining and studying the ability of red blood cells to deform. Measurements are carried out using cytodiffractometers, rheoscopes, an ectacytometer [13-17]. The principle of their measurement is the placement of red blood cells in suspension between two transparent surfaces and the creation of a counter shear flow in the flow section, simulating the conditions in the blood vessels. The flow is formed by rotating surfaces in the opposite direction. The deformation of red blood cells is measured by optical means, for example, using a photosensitive matrix.

The next method for determining the VUH of a cell is based on the use of atomic force microscopy. It is carried out by pressure of the probe of the microscope (cantilivir) on the membrane [18].

The above methods have a common and significant limitation, which inhibits their use in wide practical medicine: low accuracy and high complexity.

движения каждой клетки в суспензии и их минимальный радиус R_мин, средний радиус R и максимальный радиус R_мax через определенные интервалы времени в период воздействия электрического поля и после его отключения по истечении времени t путем видеозаписи изображения движения клеток и изменения их размера, полученные данные в цифровом виде вводят и обрабатывают в компьютере, имеющем вычислительную программу накопления и обработки данных, в результате чего определяют средние значения характеристик x_j эритроцитов для данного образца (где j - порядковый номер характеристики от 1 до 8). Причем вышеописанный процесс измерения средних скоростей

движения каждой клетки в суспензии, их минимальный R_мин, средний R_cp, максимальный R_мax радиусы и определения характеристик x_j эритроцитов проводят многократно для проб эритроцитов здоровых пациентов (в норме) и пациентов с различными видами патологий, диагноз (состояние здоровья) которых определен заранее другими методами, например иммуноферментным методом, с определением характеристик эритроцитов

(где i - порядковый номер диагноза от 1 до 7, в том числе норма и различные виды патологий: i=1 - норма, i=2 вирусный гепатит, i=3 - алкогольный гепатит, i=4 - смешанный гепатит, i=5 - вирусный цирроз, i=6 - алкогольный цирроз, i=7 - смешанный цирроз) и формируют из характеристик клеток

статистически достоверный массив данных для последующего использования его в дифференциальной диагностике заболеваний печени, а затем проводят аналогичные измерения образцов проб эритроцитов пациентов с различными видами патологий с определением лением характеристик эритроцитов x_i,j в реальном масштабе времени с последующим сравнением этих характеристик с соответствующими значениями

находящимися в базе данных компьютера, и определением показателя Δ_i близости измеряемых характеристик эритроцитов к каждому из представленных семи видов диагноза. Минимальное значение Δ_i измеряемых параметров эритроцитов человека x_j,i соответствует i-му диагнозу, который и принимают как окончательно поставленный диагноз.The closest analogue (prototype) is a method for the differential diagnosis of liver diseases (RF patent No. 2296327, IPC G01N 27/00, publ. 03/27/2007), including sampling of red blood cells, diluting them with an isotonic solution that supports the activity of red blood cell cells, with a given coefficient k, determination of the dynamic viscosity η _{w of a} suspension of red blood cells in a specified isotonic solution and transfer of a mixture of the studied red blood cells with a given cell concentration to a measuring cell, characterized in that neo homogeneous alternating electric field with a frequency f from 10 kHz to 5 MHz and an average electric field strength E ₀ in the gap between the electrodes in the range from 10 ⁴ to 10 ⁶ V / m, average speeds are measured

the movement of each cell in suspension and their minimum radius R _min , average radius R and maximum radius R _max at certain time intervals during the period of exposure to the electric field and after it is turned off after a time t by video recording the image of the movement of cells and changing their size, the data obtained in digitally enter and process in a computer having a computer program of data storage and processing, as a result of which average values of erythrocyte characteristics x _{j are} determined for a given sample (where j is serial number of the characteristic from 1 to 8). Moreover, the above process of measuring average speeds

the movements of each cell in suspension, their minimum R _min , average R _cp , maximum R _max radii and characterization x _{j of} red blood cells are carried out repeatedly for red blood cell samples of healthy patients (normal) and patients with various types of pathologies whose diagnosis (state of health) is determined in advance by other methods, for example, enzyme immunoassay, with the determination of the characteristics of red blood cells

(where i is the serial number of the diagnosis from 1 to 7, including the norm and various types of pathologies: i = 1 - normal, i = 2 viral hepatitis, i = 3 - alcoholic hepatitis, i = 4 - mixed hepatitis, i = 5 - viral cirrhosis, i = 6 - alcoholic cirrhosis, i = 7 - mixed cirrhosis) and form from the characteristics of cells

a statistically reliable data array for its subsequent use in the differential diagnosis of liver diseases, and then similar measurements of samples of erythrocyte samples of patients with various types of pathologies are carried out with the determination of the characteristics of red blood cells x _{i, j} in real time, followed by a comparison of these characteristics with the corresponding values

located in the computer database, and the determination of the indicator Δ _{i the} proximity of the measured characteristics of red blood cells to each of the seven types of diagnosis. The minimum value Δ _{i of the} measured parameters of human erythrocytes x _{j, i} corresponds to the i-th diagnosis, which is accepted as the final diagnosis.

However, the prototype method is complex and time-consuming to reproduce, requires a database of red blood cell characteristics of healthy and sick patients for comparative analysis, as well as highly qualified specialists conducting the analysis.

The technical result of the claimed invention is the creation of a simpler and less time-consuming method for the differential diagnosis of fatty liver disease of alcoholic and non-alcoholic genesis, as well as an increase in the objectivity of evaluating research indicators, regardless of the qualifications and experience of the specialist conducting the study.

The specified technical result is achieved by the fact that in the method of differential diagnosis of fatty liver disease of alcoholic and non-alcoholic genesis, which includes taking samples of red blood cells, diluting them with a solution that supports the activity of red blood cells, transferring the suspension of the studied red blood cells to a measuring cell, in which they form under the influence of an alternating voltage in the gap between two electrodes an inhomogeneous alternating electric field with a frequency of about 1 MHz, the parameters of each cell in suspension are measured in the period of exposure to the red blood cells of the electric field and after it is turned off after a time by video recording the image of the movement of cells and changing their size, the obtained data are digitally entered and processed in a computer that has a computer program for accumulating and processing data, as a result of which average values of size change are determined erythrocytes for a given sample, according to the invention, video recording of a change in the size of each red blood cell as it moves from one electrode to another during the period exposure of the electric field and after it is turned off is carried out with the receipt of several frames of the video image; Based on a computer analysis of the received video frames, software calculation is performed:

- radii (d _i ) for each individually observed red blood cell and find its arithmetic mean value (D _1,2 ) before and after turning off the electric field, the arithmetic mean value n of measuring the radius of each red blood cell before turning off the electric field from equation (1):

- сумма результатов измерений радиусов

Where:

- the sum of the measurements of the radii

i - (from 1 to n) the number of the red blood cell in the sample, the arithmetic average of n measurements for the radii of each red blood cell after turning off the electric field (2):

сумма результатов измерений радиусов

Where:

sum of radius measurement results

for the j-th red blood cell.

- the average radius (An) of the deformation amplitude for each observed red blood cell (3):

- средний радиус эритроцита под действием поля [м];Where:

- the average radius of the red blood cell under the influence of the field [m];

- средний радиус эритроцита после выключения поля [м].

- the average radius of the red blood cell after turning off the field [m].

- the average deformation amplitude (M), the standard deviation (δ) and the average error of the deformation amplitude (m) of red blood cells are, according to the known equations for the normal distribution (4):

the obtained value of the average amplitude of deformation (M) in the sample of red blood cells is the initial one - M1; then, from the same measuring chamber with a suspension of red blood cells, from 9.95 to 10.05 μl of a 0.0195-0.0205% ethanol solution are added and the mixture is exposed for about 300 seconds, and then the size change of each erythrocyte treated with ethanol is video recorded at its movement from one electrode to another during the period of exposure to the electric field and after it is turned off with the receipt of several frames of the video image; Based on the analysis of the received video frames, software calculation is performed:

- radii (d _i ) for each individually observed red blood cell and find its arithmetic mean value (D _1,2 ) before and after turning off the electric field, the arithmetic mean value n of the measurements of the radius of each red blood cell before turning off the electric field from equation (1);

- the arithmetic mean value of n measurements for the radii of each red blood cell after turning off the electric field (2);

- average radius (An) of the strain amplitude for each observed red blood cell (3);

- the average deformation amplitude (M), the standard deviation (δ) and the average error of the deformation amplitude (m) of red blood cells are found, according to well-known equations for the normal distribution (4); moreover, the average strain amplitude (M) in the sample of red blood cells after exposure to ethanol solution is (M2), then the difference M1 - M2 is determined and if this difference is statistically significant [20] and positive, then the average M1 and M2 belong to different general populations, and the conclusion is made about the presence of non-alcoholic fatty liver disease, and if this difference is significant and negative, then the average M1 and M2 belong to different general populations, and the conclusion is made about the presence of alcoholic fatty liver disease.

The method is illustrated by the following graphic materials. In FIG. 1 is a diagram of a measuring computing complex (CPI). In FIG. 2 shows a diagram of a measuring cell.

Description of a device for measuring the amplitude of deformation of red blood cells.

The measurement of the amplitude of the deformation of red blood cells is carried out using a measuring computing complex (CPI). CPI includes the following instruments and devices (Fig. 1):

- a computer (1) for recording the video image stream and processing the information stream from the video camera, calculating the characteristics of the electric field in the measuring chamber, calculating the cell parameters, storing video files and the results of calculating the cell parameters, controlling the operation (on / off) of the video recording according to a given program;

- an oscilloscope (2) Aktakom ADS2111MV for recording AC voltage on the electrodes of the measuring cell;

- mercury thermometer (3) 0-50 ° C for measuring the temperature of the cell suspension;

- generator (4) of alternating voltage G3-112 / 1;

- a microscope (5) Mikmed-6 for monitoring the cell and its reaction in the measuring chamber in response to the action of NPEP;

- object micrometers (6) OM-O, OM-P for calibrating the IVC and measuring the linear dimensions of the cell, the distance between the electrodes of the measuring chamber;

- amplifier (7) of alternating voltage G3 112/1;

- measuring cell (8);

- a visualization complex (9) MS-14 for broadcasting a cell video stream through a microscope to a computer about the dynamics of its movement in a measuring chamber;

The measuring cell (8) contains (Fig. 2): a base made in the form of a glass plate (10), on which there are metal electrodes (11) with a gap relative to each other to form a measuring chamber (12). The electrodes (11) with the measuring chamber (12) are covered with a cover glass (13).

Description of the method of differential diagnosis of fat

liver diseases of alcoholic and nonalcoholic genesis To develop a method for differential diagnosis of fatty liver disease of alcoholic and nonalcoholic genesis, the blood of volunteers was used with the approval of the Ethics Committee of the Federal State Budget Scientific Institution Scientific Research Institute of Therapy and Preventive Medicine (meeting minutes No. 122 of 11/29/2016) . All volunteers gave their informed consent to participate in the work in accordance with the Helsinki Declaration of the World Association “Ethical Principles for Conducting Scientific Medical Research with Human Participation” as amended in 2000 and the “Rules of Clinical Practice in the Russian Federation” approved by Order of the Ministry of Health of the Russian Federation of June 19, 2003 No. 266

For measurements on an empty stomach, blood with a volume of 1.95 to 2.05 ml is taken by vakutainers in a 3.65-3.85% citrate buffer in a ratio of 9: 1, and after 5 to 20 minutes a sample with a volume of 9 to 11 μl make in a 0.3 M sucrose solution with a volume of from 285 to 295 μl and mix.

The resulting cell suspension with a volume of 5 to 10 μl is introduced into the measuring chamber 12, formed by two metal electrodes 11, covered with a cover glass of size 18 * 18 mm and placed in the measuring cell 8. The distance between the electrodes is 100-150 μm. The electrodes 11 are supplied with an alternating voltage with an amplitude of U = 10 V and a frequency of f 10 ⁶ Hz and registration of the behavior of red blood cells in an electric field is carried out in automatic mode using a CPI. Under the influence of the field, red blood cells settle on the edge of the electrodes 11 and are extended along the field lines of the field (from electrode to electrode). At the fourth second after turning on the electric field, video recording of erythrocytes deposited on the faces of the electrodes is carried out in an amount of 1 to 30 video frames using the imaging complex (9). Then, immediately after shutdown, red blood cells are recorded again on the faces of the electrodes, also in an amount of 1 to 30 video frames. During this time, red blood cells return to their original state and do not change the coordinates of their location in the measuring chamber. Based on the analysis of, for example, 60 video frames, radii (di) are measured for each individually observed red blood cell and its arithmetic average value (D1,2) is found before and after the electric field is turned off. The arithmetic mean value of n measurements of the radius of each red blood cell before turning off the electric field is found from equation (1):

- сумма результатов измерений радиусовWhere:

- the sum of the measurements of the radii

i - (from 1 to n) the number of the red blood cell in the sample. Similarly, the arithmetic mean value of n measurements is found for the radii of each red blood cell after turning off the electric field (2):

сумма результатов измерений радиусов

Where:

sum of radius measurement results

for the j-th red blood cell.

This approach allows, based on a representative sample (n = 30), to determine the average radius of the deformation amplitude for each observed red blood cell (3):

- средний радиус эритроцита под действием поля [м];Where:

- the average radius of the red blood cell under the influence of the field [m];

- средний радиус эритроцита после выключения поля [м].

- the average radius of the red blood cell after turning off the field [m].

The average strain amplitude (M), the standard deviation (δ) and the average error of the strain amplitude (t) of red blood cells are found, according to the well-known equations for the normal distribution (4):

The obtained value of the average amplitude of deformation (M) in the sample of red blood cells corresponds to the initial value - M1.

Then, from the same cuvette with a suspension of red blood cells, 9.95 to 10.05 μl of 0.0195-0.0205% ethanol solution was added and its exposure was carried out for 295 to 305 seconds. At the end of the exposure time, an electric field is turned on, and red blood cells settled on the faces of the electrodes are recorded in the amount of 1 to 30 video frames. Then, immediately after shutdown, red blood cells are recorded again on the faces of the electrodes, also in an amount of 1 to 30 frames. During this time, red blood cells return to their original state and do not change the coordinates of their location in the measuring chamber. Based on the analysis of, for example, 60 video frames, the radii (di) are measured by software for each individually observed red blood cell and its arithmetic average value (D1,2) is found before and after the electric field is turned off. The arithmetic mean value of n measurements of the radius of each red blood cell before turning off the electric field is found from equation (1). Similarly, the arithmetic mean value of n measurements is found for the radii of each red blood cell after turning off the electric field according to formula (2). Then, on the basis of a representative sample (n = 30), the average radius of the deformation amplitude for each observed red blood cell is determined by the formula (3). The average strain amplitude (M), the standard deviation (δ) and the average error of the strain amplitude (m) of red blood cells are found, according to the well-known equations for the normal distribution according to formulas (4).

The average strain amplitude (M) in a sample of red blood cells after exposure to ethanol solution corresponds to (M2). During the diagnostic process, the results of the average amplitude of the deformation of the samples1 are compared in order to evaluate the reliability of the difference M1 - M2.

 If this difference is statistically significant [20] and positive, then the average M1 and M2 belong to different general populations, and it is concluded that there is a non-alcoholic fatty liver disease.

If this difference is statistically significant [20] and negative, then the average M1 and M2 belong to different populations, and a conclusion is drawn about the presence of alcoholic fatty liver disease.

The reliability of the difference between the average M1 - M2 and the diagnosis of the liver is taken, according to expression (5) and statistical indicators of the parameters of red blood cells, see table:

In a patient with fatty alcoholic liver disease, an increase in the amplitude of cell deformation occurs. In a patient with non-alcoholic fatty liver disease, a decrease in the amplitude of deformation.

Clinical examples of the use of this method of differential diagnosis of the etiology of fatty liver disease, demonstrating the possibility of effective use of the amplitude of deformation of red blood cells in clinical practice to determine the alcoholic or non-alcoholic genesis of fatty liver disease in patients with an unclear etiology of the disease.

The following are specific examples of a diagnostic method.

Example 1. Patient A., 52 years old, who came to the clinic in connection with an examination for hiring, found hepatomegaly, single vascular asterisks, and the presence of excess body weight (BMI 26.5 kg / m). On examination, the liver is enlarged, protrudes 1.5-2 cm from under the costal arch, soft elastic consistency, painless on palpation, the spleen is not palpable. According to the patient, he rarely consumes alcohol (no more than 1-2 times a month, mostly weak alcoholic drinks).

The blood composition of the patient at the time of treatment (the number of red blood cells is 4.5⋅10 ¹² / l, the hemoglobin content is 147 g / l, the color index is 0.96, the number of leukocytes is 6.2⋅10 ⁹ / l, the formula is not changed platelet count - 241⋅10 ⁹ / l, ESR - 8 mm / h, fasting glycemia - 5.9 mmol / l, total bilirubin - 15.2 μmol / l, bound (direct) - 4.2 μmol / l, free (indirect) - 11.0 μmol / L, alkaline phosphatase (ALP) - 210 U / 1 (normal to 290 U / 1), gamma-glutamyl transpeptidase (GGTP) - 41.7 U / 1 (normal to 49 U / 1), ALT - 31 U / 1 (norm up to 40 U / 1), ACT - 36 U / 1 (norm up to 37 U / 1), thymol test - 2.4 units, total cholesterol - 211 m g / dl, triglycerides - 220 mg / dl, HDL cholesterol - 43 mg / dl, total protein - 72.0 g / l, urea - 5.7 mg / dl, fibrinogen - 2.8 g / l, CRP +, prothrombin index - 81.7%). The study by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) allowed to exclude the viral etiology of the process. According to the ultrasound revealed: moderate hepatosplenomegaly, diffuse changes in the liver parenchyma of the type of steatosis, indirect signs of chronic pancreatitis.

The patient underwent a study of the electric and viscoelastic parameters of red blood cells - the amplitude of the deformation of red blood cells was determined initially and after 5 minutes of exposure with an ethanol solution. The research results showed that initially the erythrocyte deformation amplitude was reduced, at a frequency of ƒ equal to 1 MHz and a voltage of U = 10 V, it was 6.3 610 ^-6 m. After 5 minutes of exposure with ethanol solution, the erythrocyte deformation amplitude was repeatedly measured and amounted to 8.8⋅10 ^-6 m, showing an increase in dynamics of ~ 30%, which allows us to conclude that the patient has an alcoholic genesis of fatty liver disease. In this case, the difficulties in diagnosis were determined, on the one hand, by the patient’s reluctance to provide true information about the style of alcohol consumption in connection with concerns related to the transfer of information to the place of future employment. On the other hand, data from general clinical studies did not allow us to conclude that alcohol was systematically taken (all indicators were within reference values or were slightly higher, which is not surprising in the presence of alcoholic liver disease at the stage of fatty hepatosis). Using the proposed method of differential diagnosis allowed to establish the true genesis of fatty liver disease.

Example 2. Surveyed K., 60 years old, turned to an endocrinologist in connection with the presence of abdominal-visceral obesity (BMI 32 kg / m ² ). The examination revealed manifestations of the metabolic syndrome: arterial hypertension of 2 tbsp., Insulin resistance, atherogenic hyperlipidemia of type 2 B, hyperuricemia. Ultrasound examination of the abdominal organs describes a "large white liver." Biochemistry research data did not reveal changes in the level of transaminases, HGPT, bilirubin level, lipid profile changes (total cholesterol - 228 mg / dl, triglycerides - 330 mg / dl, HDL cholesterol - 37 mg / dl), increased uric acid level - 0, 41 mmol / L (N up to 0.35 mmol / L), blood glucose - 6.4 mmol / L (N up to 5.9 mmol / L). During the survey, the patient said that he drinks alcohol, but not often, finding it difficult to detail the dose and frequency.

The patient underwent a study of the electrical and viscoelastic parameters of red blood cells. The study showed that the initial values of the erythrocyte deformation amplitude were 7.9 × 10 ^-6 m. After exposure to ethanol for 5 minutes, the amplitude of cell deformation decreased by 46%, amounting to 5.4 × 10 ^-6 m. The observed dynamics allowed us to make conclusion about the presence of non-alcoholic fatty liver disease in the patient. The occasional use of alcohol in non-toxic doses had no significant effect on the development of fatty liver disease, which should be considered a manifestation of the metabolic syndrome. Thus, the above example of the implementation of the inventive method confirms the achievement of the claimed technical result, namely: the diagnosis of fatty liver disease.

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Claims (19)

A method for the differential diagnosis of fatty liver disease of alcoholic and non-alcoholic origin, including sampling of red blood cells, diluting them with a solution that supports the activity of red blood cells, transferring the suspension of the studied red blood cells to a measuring cell, in which an inhomogeneous alternating electric field with an alternating voltage in the gap between the two electrodes is formed frequency of about 1 MHz, measure the parameters of each cell in suspension during exposure to red blood cells of an electric field and after it is turned off after a period of time by video recording the image of the movement of cells and changing their size, the obtained data are digitally input and processed in a computer having a computer program for data storage and processing, as a result of which average values of changes in the size of red blood cells for a given sample are determined, characterized in that video recording changes in the size of each erythrocyte when it moves from one electrode to another during the period of exposure to an electric field and after it is turned off drive to obtain multiple frames of the video image; Based on a computer analysis of the received video frames, software calculation is performed: - radii (d _i ) for each individually observed red blood cell and find its arithmetic mean value (D _1,2 ) before and after turning off the electric field, the arithmetic mean value n of measuring the radius of each red blood cell before turning off the electric field from equation (1):

Where:

- the sum of the measurements of the radii

, i - (from 1 to n) the number of the red blood cell in the sample, the arithmetic average of n measurements for the radii of each red blood cell after turning off the electric field (2):

Where:

- the sum of the measurements of the radii

for the j-th red blood cell, - the average radius (An) of the deformation amplitude for each observed red blood cell (3):

Where:

- the average radius of the red blood cell under the influence of the field [m];

- the average radius of the red blood cell after turning off the field [m], - the average deformation amplitude (M), the standard deviation (δ) and the average error of the deformation amplitude (m) of red blood cells are found according to the known equations for the normal distribution (4):

the obtained value of the average strain amplitude (M) in the sample of red blood cells is designated as (Ml); then, from the same measuring chamber with a suspension of red blood cells, from 9.95 to 10.05 μl of a 0.0195-0.0205% ethanol solution are added and the mixture is exposed for about 300 seconds, and then the size change of each erythrocyte treated with ethanol is video recorded at its movement from one electrode to another during the period of exposure to the electric field and after it is turned off to obtain several frames of the video image; Based on the analysis of the received video frames, software calculation is performed: - radii (d _i ) for each individually observed red blood cell and find its arithmetic mean value (D _1,2 ) before and after turning off the electric field, the arithmetic mean value n of the measurements of the radius of each red blood cell before turning off the electric field from equation (1); - the arithmetic mean value of n measurements for the radii of each red blood cell after turning off the electric field (2); - average radius (An) of the strain amplitude for each observed red blood cell (3); - the average deformation amplitude (M), the standard deviation (5) and the average error of the deformation amplitude (m) of red blood cells are found according to well-known equations for the normal distribution (4); moreover, the average strain amplitude (M) in the sample of red blood cells after exposure in an ethanol solution is denoted as (M2), then the difference in values (M1 - M2) is determined and if this difference is statistically significant and positive, then the average values (M1) and (M2) are to different populations, and the conclusion is made about the presence of non-alcoholic fatty liver disease, and if this difference is significant and negative, the average values (M1) and (M2) refer to different populations, and the conclusion is made about the presence of alcoholic fatty liver disease.

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