WO2006036079A1 - Method for measuring visco-elastic characteristics of cells of biological objects, method for differential diagnosis of diffusion alcohol-induced and viral liver injuries and device for carrying out said methods - Google Patents

Abstract

The invention relates to medicine. The inventive methods and device can be used for analysing the physiological state of cells with the view of early detection of cell abnormalities in human and animal blood and for a liver-staging test. Said method consist in sampling cells, in diluting samples by an isotonic solution maintaining the vital activity of cells at a specified coefficient k, in determining the dynamic viscosity ηw of an erythrocyte suspension in said isotonic solution and in transferring the mixture of erythrocytes under examination at a specified cell concentration to a measuring cuvet (1), wherein a nonuniform electric field whose frequency ranges from 10 kHz to 5 MHz and intensity E0 ranges from 104 to 106 Volts/m is generated in a gap between electrodes connected to a supply source (4), in measuring mean movement speeds νc of each cell in the suspension and a mean radius thereof R at determined time intervals during the electric field action and after a cut-off thereof upon the expiration of a time t by video recording of cell movements and measuring the dimension thereof by means of a video camera (6) which is optically connected to a microscope (5), in inputting and processing the thus obtained digital data by means of a computer (7) provided with a data collecting and processing program, thereby determining the visco-elastic characteristics of cells.

Description

 A method for measuring the visco-elastic characteristics of cells of biological objects, a method for differential diagnosis of diffuse liver diseases of alcoholic and viral nature 5 and a device for implementing these methods

Technical field

The invention relates to the field of virology and medicine. The proposed methods and device can be used for laboratory analysis of the physiological state of cells, for example, red blood cells and white blood cells, with the aim of early diagnosis of deviations from the normal state (detection of infected or cancerous) cells in the blood of humans and animals, as well as to determine the stage of liver disease (hepatitis, cirrhosis ) and its etiology (viral, alcoholic).

15 The ability of a cell to deformation plays an important role in the process of its life. For example, red blood cells must have high elasticity in order to penetrate the capillaries. Tight contact between the walls of the capillary and the erythrocyte membrane facilitates gas exchange of oxygen and carbon dioxide, which contributes to the full provision

20 body oxygen.

A change in erythrocyte deformability is observed in certain diseases, for example, diabetes or atherosclerosis, liver diseases of various etiologies, which indicates the relevance of studying and measuring the viscoelastic characteristics of biological particles. 25 Prior Art

Differential diagnosis of diffuse liver diseases of alcoholic and nonalcoholic origin is based on a complex of clinical, biochemical, and morphological features. According to the literature, more than 26 "biological markers" of alcohol consumption are known, some of which are used in differential diagnosis liver disease. These markers include: activity of aminotransferases (ALT, ACT), their ratio, activity of gamma glutamyl transpeptidase (GGTP), alkaline phosphatase (ALP), ratio of GGTP / ALP, ferritin level, carbohydrate deficient 5 transferrin, phosphatidylethanol level, epinephrine hydrogenase, alcohol erythrocyte volume, high density lipoprotein cholesterol (HDL HDC) [S. Aradόttir, C. Moller, and C. Allip. Rhoshhatiduléthapol Formiatiop Add Degradation In Numap Ap Rat Blód.- Alsohol. 2004, v. 3 9: 8 -13]. It should be noted that in practice they use sets of these indicators because of the insufficient sensitivity or specificity of each of them taken separately. In the diagnosis of alcoholic liver lesions, the determination of carbohydrate-deficient transferrin is very promising, but its use is limited by its high cost and

15 the inaccessibility of radioimmune kits for its measurement [J. With hep, K.M. Sopigrave, P. Masaskill, J. B. Whitfield, and L. Irwig. Combipipg sarbohudré-defiсiept trаpfеrrip apd gamma-glutamultltapsfego tо iспgeаsе diagostіs assurasu for roblеm dripkіpg.- Alcohl. 2003, v. 38: 574-582].

Serum alcohol enzymes appear as early

20 signs of metabolic and structural changes during alcoholization [CK. Farrep apd K.F. Tirtop. Trait marchers for alcoholism: social utilitu. Alcohol. 1999, v. 34: 649-665] are most characteristic during the period of adaptive and compensatory changes in the organs and systems containing these enzymes. In the period of destructive changes in organs in the stage

25 decompensated enzymes representing these organs [N. Fleemap Revеw. Alcohol home detochifiсtiop: a literary review Alcohol.- 1997, v. 32: 649-656] in blood serum do not always proportionally reflect the degree of organopathology. It must be borne in mind that an increase in the activity of a number of serum enzymes is possible with liver pathology of non-alcoholic origin [S. Sherlosk. Alcoholis liver diséase. Lapett- 1995, v. 34: 227-8]. So, the activity of GGTP can increase with pathological processes in the liver, accompanied by cholestasis, with drug intoxication, pregnancy, etc. A number of authors indicate that the level and persistence of increased GGTP is significantly higher for alcoholic liver lesions [F. Duсkert, J. Johnsep, A. Ampdsep, J. Strömme, and J. 5 Morlapd Co-varioptwetweap biologic markers apd self-regrted alcohl sopsumptiop. And tweo-stud stud оf the gelatiopshir betwoop сhapgеs іn sopsumrtiop apd сhapgеs іp thе biologicіl marсеrs аmеpеmереререререререпререререререрререререререр -1992, v. 27: 545-555]. The increased activity of ACT and AJlT in a number of studies is regarded as a sign of liver damage during alcohol abuse [A. H elapder, B. Tabakoff. S resial article. BIOSMERCIAL MARCERS OF OF ALSOHOLE USE APD ABUSE: EXTRIREPS FROM THE PILOT STUDO OF THE WHO / ISBRA COMBABORATIVE PROJEST OP STATE APP TRAIT MARCHES OF ALCHHO. Alcohol. 1997, v. 32: 133-144]. Moreover, with an increased level

15 AJITs bind deeper liver damage. Some researchers believe that an increase in the ACT / AJIT ratio is more informative precisely with alcoholic liver damage [Zamip. J. et al. Thе imroptaps оf AST / ALT rаtе ip poplsoholis steatoheratitis diagposis. Arq. Gastroepterol., Jap. / Mar. 2002, vol. 39, po.l, p.22-26]. However, when conducting

20 differential diagnosis should be borne in mind that the sources of ACT are also skeletal and cardiac muscles, damage to which can lead to increased levels of ACT [S. Sherlosk. Alcoholis liver diséase. Lapett. -1995, v. 34: 227-8].

A number of authors suggest using the ratio of activity

25 GGTP / ALP for the diagnosis of alcoholic liver damage [CK. Farrep apd K.F. Tirtop. Trait marchers for alcoholism: social utilitu. Alcohol. 1999, v. 34: 649-665].

According to the literature, it is known that the level of XC JIPVP, which increases with systematic consumption of alcohol, does not increase as alcoholic liver lesions progress, which limits the use of this indicator in differential diagnosis [T.V. Chernobrovkina. Enzymopathies in alcoholism. Kiev, "Health", 1992.- p. 312].

In cases of far-reaching forms of alcoholic liver lesions, non-enzymatic methods for studying the morphological and functional safety of the liver are more indicative: determination of protein, immunoglobulin fractions and serum lipids, sedimentary samples (thymol, sublimate), indicators, excretory and metabolizing functions (plasma clearance of dyes, antipyrine index) . The mismatch of the degree of violation of the listed non-enzymatic parameters and the normal or decreasing activity of serum enzymes underlines the degree of decompensation of the organ as a result of chronic damage to alcohol.

Thus, the disadvantages of using these indicators for the differential diagnosis of liver diseases are their lack of sensitivity and specificity, as well as the high complexity, the duration of the analyzes and obtaining the source materials for a number of markers.

Known methods and devices for measuring the deformability of red blood cells by forcing through thin capillaries [WO, 02/09583, A2, 07/30/2001; SU, 1462201, 02.28.89].

However, these methods are time-consuming, give a large error in the measurements and require a large volume of the studied blood.

The closest analogue (prototype) for measuring viscoelastic characteristics is a method and apparatus for measuring viscoelastic characteristics, red blood cells in an electric field created by a high-frequency generator between electrodes in series [H. Ephelhardt, E. Sackmapp. On thе measuremet оf shear elastiс moduli apt visсositiеs оf erutrosose рlаsmа membranes by transient defеrmаtiop in high quality filters. Biorhus. J.- 1988, v. 54: 495-508]. The method includes filling a working cell volume with a suspension of measuring cells with electrodes sequentially installed in it, applying an inhomogeneous alternating electric field to the suspension, computer recognition of the maximum size of an erythrocyte attracted to the electrode, calculation of the force stretching the cell according to the model of a conducting ellipsoid.

The disadvantage of the described method and device is the low accuracy of measuring the stiffness and viscosity of the cell, due to the error in calculating the tensile force according to the model of the conductive ellipsoid and the error associated with the fact that the cell has direct contact with the surface of the electrode. This changes the physiological state of the cell membrane. The method and device have a low productivity of the process of measuring the visco-elastic characteristics of the cell. The closest analogue (prototype) of the method for the diagnosis of liver diseases is the method of differential diagnosis of diffuse liver diseases of alcoholic and non-alcoholic nature by measuring the average radius and the average corpuscular volume of red blood cells (SKOE), (MCV) [T. Wötherlipg, R. Kapitz, H. Rumf, U. Narcke, and D. Fischer. Comparison of CAGe and MAST wϊth the alcohol markers CDT, {gamma} -GT, ALAT, ASAT and MCV. Alcohol. - 1998, v. 33: 424-430], which is calculated as the ratio of the hematocrit to the number of red blood cells in 1 μl of blood. The advantage of this indicator is accessibility, a fairly high sensitivity from 29 to 95%, long-term maintenance at the same level during the period of withdrawal. The reasons for the increase in SKOE with prolonged alcoholization are the toxic effect of ethanol on myelopoiesis, impaired metabolism of B vitamins and folic acid, a change in the fluidity of erythrocyte membranes due to a change in the ratio of cholesterol / phospholipids, a change in the composition of phospholipids; increased osmotic plasma pressure. A persistent change in this indicator with alcohol damage was noted. the liver. RMSE measurement is usually carried out using a conductometric hemisphere analyzer (particle counter) based on measuring the electrical resistance of a thin capillary through which the flow of red blood cells passes. The closest technical solution (prototype) of a device for the differential diagnosis of diffuse liver diseases is a particle counter of the French company "Soulter" [FR, 2092378, 1972], containing a cuvette with an electrically conductive suspension of particles and a vertically located measuring tube with a calibrated input in the lower part a hole, the upper end of the measuring tube being connected by a pneumatic conduit to a vacuum source (a manual piston pump), an internal electrode is installed in the measuring tube, and in the cell eshny electrode. The electrodes are connected to a current source and an electronic unit for measuring and processing data. The disadvantage of these methods for diagnosing diseases and the device for its implementation is the low reliability of the differential diagnosis of diseases due to the narrow spectrum of measured parameters of human red blood cells (determining only the size of red blood cells and characteristics directly related to the size of red blood cells), as well as large errors in measuring the size of red blood cells with high degree of aggregation.

Disclosure of invention

The technical result of the invention is to increase the accuracy of measuring the viscoelastic characteristics of cells of biological objects and increase the reliability and specificity of the differential diagnosis of liver diseases and reduce the time of diagnosis by providing the ability to measure the following parameters of human red blood cells without changing the physiological state of the membrane of red blood cells: average cell radius, K _α - ratio the average polarizability α _{0 1} and sc at two frequencies, for example, 100 kHz and 1 MHz, membrane conductivity and capacitance erythrocytes, erythrocyte stiffness and viscosity, erythrocyte aggregation index, erythrocyte destruction coefficient in an electric field.

The specified technical result is achieved by the fact that in the method of measuring the visco-elastic characteristics of cells of biological objects, including sampling cells of biological objects, diluting them with an isotonic solution that supports cell activity, with a given coefficient k, determining the dynamic viscosity η _{w of a} suspension of red blood cells in the specified isotonic solution and transferring a mixture of the studied red blood cells with a given concentration of cells into a measuring cell, according to the invention, a heterogeneous shape is formed in the cell th alternating electric field with a frequency / 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 Volt / m, measure the average speed V _{c the} movement of each cell in suspension and their average radius R at certain time intervals during the period of exposure to the electric field and after it is turned off after a period of time t 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 computational The data accumulation and processing chart, as a result of which the following average values of the characteristics of the cells of biological objects, including their elastic-elastic characteristics, are determined by the formulas: cell polarizability

(where ^ is the dynamic viscosity of the fluid, R is the average radius of the cell, average velocity v _{c the} movement of each cell in suspension,

^ is the intensity of the external electric field, ε _{o is the} dielectric constant of the vacuum); force (F _el ), a stretching cell, in the direction of the electric field vector:

stiffness (s) of the cell:

a .. - ε _p E ² s =

A - R - Ax

where: the strain Δx after turning off the electric field; viscosity (ή _c ) cells:

where: the strain Δx after turning off the electric field, t-time after turning off the electric field, ^ is the strain value Ax after turning off the electric field.

Thus, to ensure high accuracy in measuring the viscoelastic characteristics of a biological cell, the optimal frequency, amplitude and duration of exposure to an electric field are selected under the condition of a given value of cell deformation. As a rule, for each type of biological cell, there are critical frequency and field strengths at which the deformation efficiency is maximum, which ensures high accuracy in measuring the viscoelastic characteristics of the cell.

The specified technical result is also achieved by the fact that in the method of differential diagnosis of liver diseases, which includes taking samples of red blood cells, diluting them with an isotonic solution that supports the vital functions of red blood cells, with a given coefficient k, determining the dynamic viscosity η _{w of a} suspension of red blood cells in a specified isotonic solution and transferring a mixture of the studied red blood cells with a given cell concentration to a measuring cell, according to the invention, an inhomogeneous alternating electric field with a frequency / from 10 kHz to 5 MHz and an average intensity E ₀ electric field in the gap between the electrodes in the range of 10 ⁴ to 10 ⁶ V / m, measured average traffic speed V _c of each cell in the slurry and minimum radius R _mun mean radius R and ma maximum DUTY radius R _MAX at regular intervals during the action of the electric field off and after the time t by a video image motion of cells and changes their size, the data in digital form is introduced and treated in a computer having a data acquisition and processing computer program, as a result, determine the average values of the characteristics X _{j of} red blood cells for a given sample (where j is the serial number of the characteristic from 1 to 8):

Xj = R is the average radius of the cell, calculated by the formula:

F _mp = 6 - π - η _w - R - V _e

where P _mp is the force of viscous friction on the liquid side; 77w ~ D is the ^individual viscosity of the liquid, R is the average radius of the cell, v _c is the average speed of each cell in suspension;

X ₂ = K _a = α _o .i / αь is the ratio of the average polarizability α _{c = 0.} i and α _{c = 1} at two frequencies, for example, 100 kHz and 1 MHz, which (α _c = _O l and α _{c =} i) are calculated from the condition that the electric field and the viscous friction force are equal according to the formula:

24 - π - η _w - R - v _c ac = ε _o - grad (E _o ² ) where: / 7 _W is the dynamic viscosity of the fluid, R is the average radius of the cell, the average speed v _{c the} movement of each cell in suspension,

^ -strain of the external electric field, εo-dielectric constant of the vacuum; X3 = σ _m = 0.00003 • Kl + 0.0001 • K _a ² + 0.0001 ^• K _a + 0.00005 - membrane conductivity, where: f _eg - the equilibrium frequency is calculated by the formula:

где /о_{, /}-нижняя частота, на которой измеряется поляризуемость аоj, /_/-верхняя частота, на которой измеряется поляризуемость α_/.

where / o _{, / is the} lower frequency at which the polarizability aoj is measured, / _{/ is the} upper frequency at which the polarizability α _/ is measured.

X _f = C _1n = is the capacity of the cell membrane,

where: σ is the electrical conductivity of the solution in which the measurements are made.

X ₅ = c = - - - - - -. - stiffness of the cell membrane, where: 4 - R - Ax ^F

Ax is the magnitude of the deformation of the cell after turning off the electric field; c - t Xl ⁼ Lr cell viscosity, where: c 6 - π - R - In (Ax / A)

/ - time after turning off the electric field, Λ - strain value Δх at the moment of turning off the electric field;

N d; ₇ = K _ag = - - - erythrocyte aggregation index, where:

N _d is the number of adherent cells in the field of observation of the microscope; N ₀ is the total number of cells in the field of observation of the microscope;

N -N χ ₈ ⁼ K ₁₁ . = - - .- cell destruction coefficient, where:

^ o

N ( _ts is the number of cells remaining in the field of observation of the microscope after destruction; N ₀ is the total number of cells in the field of observation of the microscope; moreover, the above described process of measuring average speeds v _{c the} movement of each cell in suspension, their minimum R _moon , average R _cp maximum R _max radii and characterization X _{j of} red blood cells is carried out repeatedly for red blood cell samples of healthy patients (normal) and patients with various types pathologies, the diagnosis (state of health) of which is determined in advance by other methods, for example, enzyme immunoassay, with the determination of the characteristics of red blood cells X _J ⁰ ₁ (where i is the serial number of the diagnosis from 1 to 7, including the norm and various types pathologies: i-1 - normal, i = 2 viral hepatitis, i = 3 - alcoholic hepatitis, i = 4 - mixed hepatitis, i = 5 - viral cirrhosis, i = b

- alcoholic cirrhosis, i = 7 - mixed cirrhosis) and form from data

X _J ⁰ _{1 is a} statistically significant array for its subsequent use in the differential diagnosis of liver diseases, and then similar measurements of samples of red blood cells of patients with various types of pathologies are carried out with the characterization of red blood cells Xμ in real time, followed by comparison of these characteristics with the corresponding values of _{J J} ° _t , located in the computer database and the determination of the indicator Δ, the proximity of the measured characteristics of red blood cells to each of the seven types diagnosis:

J = s

^A. = (∑ ^K j - ( ^χ _J , - ^χ _J ⁰ , Y ^{Д for} / = 1-7,

значение у-ого параметра из всех x°_a параметров, соответствующих всем i- ым диагнозам; MMH_j (X_J ⁰ ₁ ) - минимальное значение у-ого параметра из всехwhere K _j - normalizing diagnostic coefficients, which are calculated by the formula:

the value of the y-th parameter from all x ° _a parameters corresponding to all i-th diagnoses; MMH _j (X _J ⁰ ₁ ) - the minimum value of the y-th parameter of all

соответствует /-тому диагнозу, который и принимают как окончательно поставленный диагноз.X _j about ₁ parameters corresponding to all the rth diagnoses, and the minimum value of Δ ,, measured parameters of human red blood cells

corresponds to the / -th diagnosis, which is accepted as the final diagnosis.

The specified technical result is also achieved by the fact that in the device for measuring the viscoelastic characteristics of bioobject cells and for differential diagnosis of diffuse liver diseases, including a transparent measuring cell, in which electrodes are connected to a power source, as well as a measuring unit, according to the invention, the power source represents a variable voltage generator, and the measuring unit contains a microscope, optically coupled to a measuring cuvette and image analysis system for measuring the speed of red blood cells containing a video camera, optically coupled with a microscope, and a computer connected to the camera. Moreover, the electrodes in the measuring cell are installed with a gap sufficient for the formation of an average electric field in it in the range from 10 ⁴ to 10 ⁶ Volt / m.

Under conditions of an inhomogeneous alternating electric field (NPEP), the mobility of red blood cells in suspension is tested. The experiments showed that red blood cells have significant mobility in an alternating inhomogeneous electric field. In a weakly conductive glucose solution, red blood cells normally progressively move to a region with a high electric field strength at a high frequency ("1-5 MHz) of the electric field. At a lower frequency of the electric field ("10-100 kHz), red blood cells move to the area with low electric field strength. In addition, it was found that red blood cells with liver pathology lose their ability to move in NPEP. The fundamental difference in the mobility of red blood cells in normal and pathological conditions and in NPEP is the basis of the invention. The ratio of erythrocytes moving at different speeds in NPEP is associated with the degree of liver pathology. The invention is illustrated by the following graphic materials. In FIG. 1 shows a block diagram of an automated installation for implementing the proposed method for measuring the visco-elastic characteristics of cells of biological objects and a method for differential diagnosis of diffuse liver diseases. In FIG. 2 shows a diagram of a measuring cell. In FIG. 3 shows the design of an existing automated installation for implementing the proposed method for measuring the visco-elastic characteristics of cells of biological objects and a method for differential diagnosis of diffuse liver diseases.

Embodiments of the invention

A device for measuring the viscoelastic characteristics of biological cells and for the differential diagnosis of diffuse liver diseases includes a collapsible optically transparent measuring cell 1 (Fig. 2), in which metal electrodes 2 and 3 are located, connected to an electric power source 4, as well as a measuring unit. The power source 4 is an alternating voltage generator, and the measuring unit (Fig. 1) contains a microscope 5, optically coupled to a measuring cell 1 and an image analysis system for measuring the speed of test cells, containing a video camera 6, optically connected to a microscope 5, and a computer 7 connected to the video camera 6. Computer 7 contains a specialized image processing program. Measuring cuvette 1 is placed on a movable table 5. Moreover microscope electrodes 2 and 3 in the measuring cell 1 fitted with a clearance sufficient to form therein the average electric field in the range of from 10 ⁴ to 10 ^b Volt / m. The gap between the electrodes 2 and 3 in the experimental design of the cuvette 1 is set within 50-100 microns, and the thickness of these electrodes is 0.2-2 microns. The design of the experimental operating installation (Fig. 3) additionally provides for an oscilloscope 8 connected to electrodes 2 and 3 of the measuring cell 1 for monitoring electrical parameters, as well as a monitor 9 connected to a digital video camera 6.

The device operates as follows. The measuring cuvette 1 is mounted on a movable table of the microscope 5 and the indicated cuvette 1 is fixed on it. A sample of a cell suspension with a known degree of dilution is introduced into the measuring cuvette 1. The electrodes 2 and 3 of the measuring cuvette 1 supplied voltage (not more than 10 volts) from the source 4 (generator) AC voltage between which is formed an average electric field intensity in the range of from 10 ⁴ to 10 ^b Volt / m. Using a video camera 6, the dynamics of individual cells in the measuring cell 1 is recorded. From a video camera 6, a video signal of the dynamics of cell movement is fed to a computer 7 (with a specialized image processing program), where data are processed and cell characteristics of biological objects, including their viscoelastic, are calculated characteristics used, for example, for the differential diagnosis of diffuse liver diseases.

The advantage of the device for measuring the viscoelastic characteristics of a cell of biological objects using a high-frequency electric field compared to existing analogues is that it provides an increase in the accuracy of measuring the viscoelastic characteristics of a cell at the same time both by changing the amplitude and by the frequency of the alternating voltage on the electrodes in the cell for measurements, and reduction of time for one measurement. Compared with the prototype, in which the deformation of the cells of biological objects is ensured only by changing the amplitude of the pulse voltage at the electrodes in the proposed device, by changing both the amplitude and frequency of the alternating voltage at the electrodes, a higher accuracy of measuring the viscoelastic characteristics of the cell is provided A method for measuring the visco-elastic characteristics of cells of biological objects and a method for differential diagnosis of diffuse diseases of the liver are implemented as follows.

1st stage of measurements and calculations. It relates both to a method for measuring the characteristics of cells of biological objects, including viscoelastic characteristics, for example red blood cells, and to a method for differential diagnosis of diffuse liver diseases.

For this, preliminary sampling of cells of biological objects, for example, blood samples, is carried out. Samples are diluted with a given coefficient k, and the mixture of the studied material samples with a given cell concentration C k is transferred _to measuring cell 1. In cell 1, an inhomogeneous alternating electric field is formed with a frequency of 10 kHz to 5 MHz and an average electric field strength in the gap between the electrodes in in the range from 10 ⁴ to 10 ⁶ Volt / m by applying voltage (not more than 10 Volts) to the electrodes 2 and 3 from the source 4 (generator) of power supply. Next, measure the average speed v _{c the} movement of each cell in suspension and their minimum radius R _mun , the average radius R and the 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 cell movement and changes 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 characteristics of X _j cells are determined biological objects, including the elastic-elastic characteristics of red blood cells, for a given sample (where j is the serial number of the characteristic from 1 to 8).

Under conditions of inhomogeneous electric fields, the average speed of translational movement of the cell v _c is determined by the equality of the average value of the force from the side of the inhomogeneous electric field <F _ef > = a _c - ε ₀ - grad {El) (1) where: a _c is the polarizability of the cell, EO is the intensity of the external electric field, ε ₀ is the dielectric constant of the vacuum. The force of viscous friction on the side of the liquid P _mp,:

P _mp = 6 - π - η _w - R - V _c (2) where ^ is the dynamic viscosity of the fluid, R is the average radius of the cell, which is the first diagnostic parameter X ₁ ^.

The polarizability of the cell is calculated from the condition of equality of force from the electric field and the force of viscous friction according to the formula

By calculating the ratio of the average polarizability at two frequencies (100 kHz and 1 MHz), the second of the diagnostic parameters Xμ

The third diagnostic parameter is the membrane conductivity, which is determined by the formula: x ₃ .i = σ _m = 0.00003 - A ^ + 0.0001 - U- ^ + 0.0001 K _a + 0.00005 (4)

The equilibrium frequency is calculated by the formula:

где fоj-няжняя частота, на которой измеряется поляризуемость a_Oj, /у-верхняя частота, на которой измеряется поляризуемость α_/. Четвертым диагностическим параметром является емкость мембраны, которая вычисляется по формуле:

σ -электропроводность раствора, в котором проводятся измерения. Сила, со стороны электрического поля, растягивающая клетку, вычисляется по формуле:

where foj is the gentle frequency at which the polarizability a _Oj is measured, / y is the upper frequency at which the polarizability α _/ is measured. The fourth diagnostic parameter is the membrane capacity, which is calculated by the formula:

σ is the electrical conductivity of the solution in which the measurements are made. The force, from the side of the electric field, stretching the cell, is calculated by the formula:

_^ a _c - ε ₀ - E ₀ ² A - R

Cell stiffness (c) is the fifth diagnostic parameter of the cell (viscoelastic characteristic of the cell) and is calculated depending on the amount of deformation Ax according to the formula:

(* „S ₀ - E ₀

X ₅ J = C = (8)

A R - Ax

After turning off the electric field, the original shape of the cell is restored. In this case, the strain value Ax decreases exponentially:

Ax = A - exp (-χ • t). (9) where is the t-time after turning off the electric field,, 4 is the strain value Ax at the moment the electric field is turned off.

The rate of change of the strain γ depends on the stiffness and viscosity of the cell η _c :

γ = ~ * 6 • π - ^" η _c - R О ^' ( ^{1 0} ) ^y Measurement of the dependence of cell deformation on time determines the viscosity of the cell, which is the sixth diagnostic parameter of the cell (visco-elastic characteristic of the cell):

By measuring the ratio of the number of adherent cells N _rf to the total number of cells N ₀ in the field of observation of the microscope, the erythrocyte aggregation index is determined, which is the seventh diagnostic parameter: v V - ¹ ^ X7, v ^{= K} ag ~ (12)

N _n

By measuring the ratio of the number of N ^ cells remaining in the field of observation of the microscope after destruction to the total number of cells N ₀ in the field of observation of the microscope, the cell destruction coefficient is determined, which is the eighth diagnostic parameter:

_ _fr N ₀ - N ^

* ". Ι -л _Л - - • (13) i ^V 0

2nd stage of measurements and calculations. It applies only to the method of differential diagnosis of diffuse liver diseases. The process described above (stage 1) measures the average speeds v _{c of} movement of each cell in suspension, their minimum R _moon , average R _cp maximum R _max radii and characterization x _{j of} red blood cells on each frame of the video clip repeatedly for red blood cell samples of healthy patients (normal) and patients with various types of pathologies, the diagnosis (state of health) of which was determined in advance by other methods, for example, enzyme immunoassay, determination of the characteristics of red blood cells x ° ₍ (where i is the diagnosis serial number from 1 to 7, including the norm and various types of pathologies: i = l - 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).

Then a statistically reliable data array is formed from the characteristics x _J ° _t , which is stored in the computer memory for subsequent use in the differential diagnosis of liver diseases.

Conduct similar measurements of samples of red blood cells of patients with various types of pathologies with the determination of the characteristics of red blood cells Xμ in real time with subsequent comparison of these characteristics with the corresponding values of x °. located in the computer database and the definition of the indicator Δ _; the proximity of the measured characteristics of red blood cells to each of the seven types of diagnosis presented:

7 = "

Δ, = (∑KJ - ( _{XJ <I} - x °,) ² for ι = l-7, (14)

where K _j - normalizing diagnostic coefficients, which are calculated by the formula:

K ₁ = - r-, (15), where: MAKC ₁ (X ⁰ ,,) -

¹ MAX _} (x) ^ - MIN ^ x) At ^{Л h> the} maximum value of the y-th parameter of all x ° parameters corresponding to all z-th diagnoses; MMH _j (X _J ⁰ ₁ ) - the minimum value of the / -th parameter of all x _j ° ₄ parameters corresponding to all z-th diagnoses.

Normalizing diagnostic coefficients K _j allow one to compare in dimensionless form the measured parameters having different dimensions, as well as bring the values of these parameters to values close to unity.

Next, choose the minimum value of Δj, the measured parameters of human red blood cells X _{j; i,} corresponding to the / -th diagnosis, which is accepted as the final diagnosis.

Example 1. Determination of the viscoelastic characteristics of red blood cells

The experiments were carried out on the setup shown in FIG. 3.

The cuvette 1 is made in the form of two glass plates on which electrodes 2 and 3 are parallel formed by photolithography. To increase the life of the electrodes, they are coated with a dielectric layer. The distance between the plates is 50 microns. A sample of human red blood cells is diluted in a 5% glucose solution in a ratio of 1: 1000 to obtain a suspension of red blood cells until a concentration is reached erythrocytes, for example, 1 million / ml with a dynamic viscosity of the suspension η _w> equal to 1.2-10 ^"3 groin. After the cell suspension comes to rest, an alternating voltage is applied to the metal electrodes 2 and 3 of the measuring cell 1 (not more than 10 V.) The flow rate is 50 5 μm / s.The optimal frequency of the electric field is 1 MHz, at which the deformation of human red blood cells is most pronounced, and the amplitude of the electric signal is 10 V. The experiments showed that for each of the studied cell types there is an optimal The values of frequency and amplitude of the electric field at which the maximum th degree of deformation.

Next, measure the average speed v _{c the} movement of each cell in suspension and their minimum radius R _mun , the average radius R and the maximum radius R ^ at certain time intervals during the period of exposure to the electric field and after it is turned off after 15 times t by recording the image of the movement of cells and resizing the received data in digital form is introduced and treated in a computer having processing and data storage processing program, thereby determining the average characteristic values X _j cells iologicheskih objects, including a resilient, elastic characteristics erythrocytes, for a given sample (where j - sequence number of features from 1 to 8). Computer data for clarity are presented in table 1.

Comparison of experimental data (table 1) obtained by measuring the stiffness and viscosity of red blood cells and recalculated by

25 formulas (1-12) and the formulas used in the prototype allow us to conclude that taking into account the polarizability coefficient and other physical parameters proposed in the inventive method reduces the spread of data within one sample in terms of stiffness with

60% (prototype) to 43.3% (the inventive method), and in viscosity from 97% zo (prototype) to 42.9% (the inventive method). Table 1

Comparative data on the visco-elastic characteristics of red blood cell cells obtained by the claimed method and the prototype

Пример 2. Формирование статистического массива данных для дифференциальной диагностики диффузных заболеваний печени

Example 2. The formation of a statistical data array for the differential diagnosis of diffuse liver diseases

To generate a statistical data array for the differential diagnosis of diffuse liver diseases, measurements of the characteristics of red blood cells x °, were carried out. 432 patients in Novosibirsk with various diagnoses. Diagnoses are made by laboratory enzyme-linked immunosorbent assays and documented by medical records. Among 432 men (from 33 to 64 years old): 300 - with liver pathology of alcoholic and viral origin and 132 healthy.

The method of preparing red blood cell samples and the measurement process in cuvette 1 during the period of exposure to the electric field and after it is turned off after a time t by recording the image of the movement of cells and changing their size is described in Example 1. After measuring the characteristics x ° _. erythrocyte cells (where i is the diagnosis serial number from 1 to 7, including the norm and various types of pathologies: i — 1 is the norm, i = 2 is viral hepatitis, i = 3 is alcoholic hepatitis, i = 4 is mixed hepatitis, i = 5 - viral cirrhosis, i = 6 - alcoholic cirrhosis, i = 7 - mixed cirrhosis) calculate the characteristics of red blood cells according to formulas (1-13). A statistically reliable data array is formed from them, which 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 the characteristics x °, erythrocyte cells are determined. A computer fragment of the averaged characteristics of x °, erythrocytes of patients in this statistical array is presented in table 2 for clarity. table 2

Experimental data on a method for differential diagnosis of diffuse liver diseases in liquid biomaterial

The obtained statistically reliable data array is used later in the differential diagnosis of diffuse liver diseases. Example 3. Differential diagnosis of diffuse liver disease in real time

Example 3.1 Patient B., as a result of measurements of red blood cell characteristics, received the following analysis results

(line 2, table 3). Then, according to formulas (13-15), the indicator Δ is calculated, the proximity of the measured characteristics of red blood cells to each of the seven types of diagnosis presented. The calculation results are presented in table 3 (lines 4-10). The sum of the deviations of the characteristics of the patient's red blood cells from the tabular characteristics of the corresponding Table 3 shows the different diagnoses. Table 3 shows the minimum deviation of the patient erythrocyte characteristics from the table characteristics Δj in the case of 0.13, which corresponds to the diagnosis N ° l - normal.

Table 3 The results of studies of the characteristics of the red blood cells of the patient

Example 3.2. Patient C, as a result of measurements of the characteristics of his red blood cells, received the following analysis results

(see row 2 in table 4. Then, the indicator is calculated by formulas (13-15)

Δj proximity of the measured characteristics of red blood cells to each of the seven types of diagnosis. The calculation results are presented in table. 4 (lines 4-10). The sum of deviations of the patient’s erythrocyte characteristics from tabular characteristics corresponding to different diagnoses is presented in column 10 of the table. 4. The minimum value of the deviation Δj of the patient erythrocyte characteristics from the tabular characteristics Δj is equal in this case to 0.13, which corresponds to a diagnosis of N ° 7 (cirrhosis of mixed etiology).

Table 4 The results of studies of the characteristics of the red blood cells of the patient

From tables 3 and 4 it can be seen that the advantage of the proposed methods compared to the existing ones lies in the fact that they provide a measurement of the characteristics of biological object cells and differential diagnosis of diffuse liver diseases with significant savings in time and material costs. The analysis time is reduced to 3 minutes, which is more than 100 times less compared to the prototype method.

Industrial applicability. The proposed device can be manufactured in a small enterprise and industrial production using standard equipment, modern materials and technology, which implements a method for measuring the visco-elastic characteristics of red blood cells and a method for differential diagnosis of diffuse liver diseases.

Claims

Claim 1. A method for measuring the visco-elastic characteristics of cells of biological objects, including sampling cells of biological objects, diluting them with an isotonic solution that supports cell activity with a given coefficient k, determining the dynamic viscosity Tj _{w of a} suspension of red blood cells in the specified isotonic solution and transferring the mixture of the studied red blood cells with a predetermined concentration of cells in the measuring cell, characterized in that an inhomogeneous alternating electric field with a frequency of / from 10 kHz d is formed in the cell 5 MHz and average intensity E ₀ of the electric field in the gap between the electrodes in the range of 10 ⁴ to 10 ⁶ V / m, measured average velocity v _c movement of each cell in the slurry and their average radius R at regular intervals during the action of an 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 obtained data are digitally input and processed in a computer having a computer program for data storage and processing, as a result of which о determine the following average values of the characteristics of cells of biological objects, including their elastic-elastic characteristics, according to the formulas: polarizability of cells

(where ^ is the dynamic viscosity of the fluid, R is the average radius of the cell, average velocity v _{c the} movement of each cell in suspension, ^ is the intensity of the external electric field, ε _{o is the} dielectric constant of the vacuum); force (F _el ), stretching the cell, in the direction of the electric field vector:

stiffness (s) of the cell: 5

where: the amount of deformation Ax after turning off the electric field; viscosity (ή _c ) cells:

where: the strain value Ax after turning off the electric field, t-time after turning off the electric field, ^ is the strain value Ax after turning off the electric field. 2. A method for the differential diagnosis of liver diseases, including sampling of red blood cells, their dilution isotonic 15 a solution that supports the vital 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 concentration of cells to a measuring cell, characterized in that 20 inhomogeneous alternating electric field with a frequency / of 10 kHz to 5 MHz and average intensity EQ electric field in the gap between the electrodes is in the range of 10 ⁴ to 10 ^b Volt / m was measured average speed V _{c the} movement of each cell in suspension and their minimum radius R MUIt) the average radius R and the 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 movement of cells and changing their size, the obtained data are digitally input and processed in a computer having a computer storage program and data processing, as a result of which determine the average values of the characteristics X _{j of} red blood cells for a given sample (where j is the serial number of the characteristic from 1 to 8): X ₁ = R is the average radius of the cell, calculated by the formula: P _mp = 6 ^• π - η _w ^• R ^• V _e where P _mp is the force of viscous friction on the liquid side; ^ is the dynamic viscosity of the fluid, R is the average radius of the cell, V _c is the average velocity of each cell in suspension; Xj = K ₀ = αо.i / tti, is the ratio of the average polarizability α _{c =} o _. i and α _{c = 1} at two frequencies, for example, 100 kHz and 1 MHz, which (α _{c = O} l and α _c = i) are calculated from the condition that the electric field and the viscous friction force are equal according to the formula: 24 - π - η _w - R - v _c ac = ε ₀ ^• grad (E ₀ ) where: 7 / _and , is the dynamic viscosity of the liquid, R is the average radius of the cell, the average speed v _{c the} movement of each cell in suspension, £ O-tension of an external electric field, ε _o- dielectric constant of vacuum; x ₃ = σ _m = 0.00003 ^• K "l3 +. r 0 \. n0л0n0i1 • K v _a 2 ^ι + 0.0001 • K _a + 0.00005 - membrane conductivity, where: f _eg - the equilibrium frequency is calculated by the formula:

where f _{Oi / is the} lower frequency at which the polarizability a _O j is measured, / _{/ is the} upper frequency at which the polarizability α _y is measured. R σ - ^X ₄ ⁼ C _1n = is the capacity of the cell membrane, where: σ is the electrical conductivity of the solution in which the measurements are made. Xh = c = - - - - -. - the stiffness of the cell membrane, where: 4 R - Ax ^Y Ax is the magnitude of the deformation of the cell after turning off the electric field; s - t Xo = U _r = cell viscosity, where: ⁶ ' ^c 6 π - R \ n (Ax / A) t-time after turning off the electric field, L-value of the strain Ax at the time the electric field is turned off; N x ₇ = K _ag = - - - the index of aggregation of red blood cells, where: N _d is the number of adherent cells in the field of observation of the microscope; N ₀ is the total number of cells in the field of observation of the microscope; N - N χ ₈ ⁼ K _ώ = - - .- cell destruction coefficient, where: N _ds is the number of cells remaining in the field of observation of the microscope after destruction; N ₀ is the total number of cells in the field of observation of the microscope; moreover, the above process of measuring average speeds v _{c the} movement of each cell in suspension, their minimum R _moon , average R _cp maximum R _max radii and characterization X _{j of} red blood cells is carried out repeatedly for red blood cells of healthy patients (normal) and patients with different types pathologies, the diagnosis (state of health) of which is determined in advance by other methods, for example, enzyme immunoassay, with the determination of the characteristics of red blood cells X _J ⁰ _1I (where i is the serial number of the diagnosis from 1 to 7, including the norm and various types pathologies: / = / - 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 are formed from the characteristics of JC ° cells _(a statistically significant data set for subsequent use in differential diagnosis 5 liver diseases, and then carry out similar measurements of samples of erythrocyte samples from patients with various types of pathologies with the determination of the characteristics of red blood cells X _{j; i} in real time with the subsequent comparison of these characteristics with the corresponding values of x ° _t , dressed in a computer database and determining Δ, the proximity of the measured characteristics of red blood cells to each of the seven types of diagnosis presented: Δ, = (| χ ^• (* ,,, - <,) ² For / = 1-7, where K _j - normalizing diagnostic coefficients, which 15 are calculated by the formula: K = - 7—, where MAKC ₁ (X ⁰ ,,) is the maximum 'MAX, ",) -MIN, (*",)' ^{D l "} ' the value of the y-th parameter of all x _J ° _t parameters corresponding to all _r- th diagnoses; MHH _j (X _{J ^0, 1)} - the minimum value y ^'th parameter from all X _J ⁰ ₁ parameters corresponding to all the i-th diagnoses, wherein the minimum value Δ 0 ₁₅ Human erythrocyte parameters measured hμ corresponds to g-diagnosis, which taken as a definitive diagnosis. 3. A device for measuring the viscoelastic characteristics of biological object cells and for differential diagnosis of diffuse diseases of the liver 5, including a transparent measuring cell (1), in which electrodes (2) and (3) are located, connected to the power supply (4), as well as a measuring unit characterized in that the power source (4) is an alternator voltage, and the measuring unit contains a microscope (5), optically connected to a measuring cell (1) and an image analysis system for measuring the velocity of red blood cells, containing a video camera (6), optically connected to a microscope (5), and a computer (7), i connected to the camcorder (6). 4. The apparatus of claim. 3, characterized in that the electrodes (2) and (3) in the measuring cell (1) are mounted with clearance sufficient to form therein the average electric field in the range of from 10 ⁴ to 10 ^b Volt / m .