Method for blood cell analysis for glucose content

FIELD: medicine.

SUBSTANCE: patient's arterial whole blood is sampled. The blood sample is sounded in a dish with laser at long wave within the range 500-1100 nm spatially focused in a separate blood cell selected from erythrocytes or leukocytes. It is followed with measuring a Raman spectrum in the spectral range with Stockes frequency shift within 100 cm-1 to 3200 cm-1 from a set blood cell volume determined by a confocal volume described by an interfaced microdiaphragm diameter. The Raman spectrum of glucose, haemoglobin and water in the concentrations typical for their content in a blood cell are pre-measured. The Stockes spectral components are selected in the Raman spectrum to match the typical resonant oscillations of glucose molecules and to fall to a minimum of the Stockes spectral components of the Raman spectra of haemoglobin and water. The amplitude of the Stockes spectral components of glucose is measured and related to the amplitudes of the Stockes spectral components of a calibration curve of the Raman spectra of glucose generated in the known concentration of a glucose solution to measure the glucose content in a separate blood cell.

EFFECT: invention enables noninvasive glucose measurement inside a separate alive blood cell selected from erythrocytes or leukocytes.

5 dwg

 

The invention relates to the field of biomedical technologies, in particular for laboratory diagnosis and determination of glucose in plasma, whole blood and shaped formations of blood on the basis of the analysis of the changes of the Raman spectra of glucose in individual living cells and blood plasma using a confocal laser scanner. This method allows to estimate the glucose is not only in blood but also within individual erythrocyte or leukocyte, which is especially important for the diagnosis and monitoring of diabetes and the degree of influence of insulin on the entry of glucose across plasma membrane of a cell.

There is a method of determining the concentration of glucose in human blood based on measuring the total electrical resistance of the skin or one of the components of the total electric resistance of the skin (see RF patent №2230485, IPC AV 5/053). The method provides a more accurate determination of glucose concentration in human blood. Measure the total electrical resistance of the skin or one of the components of the total electric resistance of the skin, and the concentration of glucose in the blood is determined from the expression:where G(t) - determine the value of glucose concentration in the blood at time t; G0- the value of glucose concentration in the blood at the initial instant the time of the measurement process; q - value, which characterizes the ability of the organism to maintain homeostasis with respect to the concentration of glucose in the blood; G1=G0-q; a0- coefficient characterizing the relationship between values of the total electric resistance or the values of the components of the total electric resistance of the skin and the concentration of glucose in the blood of a particular person; a1- coefficient taking into account the variability of external factors and characteristics of the body of a particular person; N(x) is the normalized measured values of the total electric resistance of the skin or components of the total electric resistance of the skin, these values of q, a0and a1determine at the preparatory stage, during which over time So simultaneously measure the electrical resistance of the skin or components of a complete electrical resistance of the skin and the concentration of glucose invasive method, and the above variables q, a0and a1determined by approximation of the dependence of the concentration of glucose in blood obtained invasive method for the above-mentioned dependence of G{t), the time T is chosen to be sufficient for you to commit changes in the concentration of glucose in the blood associated with natural diurnal cycle changes caused by artificial or what about, for example, nutrition, physical activity, injection drug glucose or insulin.

This method potentially has low sensitivity to the determination of the glucose concentration, as indirect uses a calculation formula, which includes hard-to-detect parameters q and0and a1that take into account the relationship of the concentration of glucose with the electrical parameters of the skin, which are strongly dependent on the ionic composition of the liquid and sweating, with a very large variation in the sensed individuals. Furthermore, the method does not allow to measure the glucose content in the blood cells.

There is a method of determining the concentration of glucose in human blood and continuous monitoring of glucose concentration in human blood (see RF patent №2342071, IPC AV 5/053). How is that measured the electrical transfer function by means of two pairs of four-electrode sensors mounted on the surface of the body, and the first couple of docked along the main blood vessels, mainly of the limbs, and the second pair of electrodes fixed in the same place orthogonal to the first, continuously measure the electrical transfer function of not only the surface of the skin and subcutaneous tissues, then process the measuring electrode of the sensor at a pre-otka abravanel mathematical models, moreover, the model is calibrated by comparing the results of the proposed method for determination of glucose in human blood and any other known method for determination of glucose in human blood, and then calculate the concentration of glucose in human blood obtained by the author of the dependency.

The method should have low sensitivity to determine the concentration of glucose, as glucose is electrically neutral. Its concentration in the blood is three orders of magnitude smaller than the electrolytes in the blood and in tissues. Furthermore, the method does not allow to measure the glucose content in the blood cells.

There is a method of determining glucose in whole blood (see RF patent №2050545, IPC G01N 33/48). The inventive sample of whole blood is introduced into contact with the reagent, which through a chemical reaction with glucose in the sample leads to detectivemisa the change in the concentration of the dye, the value of which is determined as a measure of glucose in the sample. The sample is initially being introduced in undiluted microcuvette having at least one cavity for receiving the sample. The cavity inside is pre-processed by the reagent in dry form and the chemical reaction proceeds in this cavity. The active components of the reagent contain a hemolysis agent to influence glucose in the blood cells of the sample, for ODA is dividing the total glucose, as well as the agents that take part in chemical reactions and providing a change in the concentration of dye in the wavelength range outside the range of the absorption of hemoglobin. The absorbance measurement is performed in the above wavelength range directly on the sample in the cell.

This method has low sensitivity, as well as the characteristic IR absorption spectrum of glucose, where not influence the absorption of hemoglobin, corresponds to the region of 1.5 microns and overlaps with the resonance peak of water absorption in the region of 1.45 μm, and the concentration of water is three orders of magnitude more. In addition to measurement, it is necessary to cause hemolysis of red blood cells to measure the absorption, and therefore no way to measure the glucose content in the blood cells.

There is a method of determining the concentration of glucose in human blood (see U.S. patent No. 7,353,055, IPC AV 5/00)based on interferometric measurement of phase shift of the coherent beams in the sensing of glucose in the blood.

However, this method has a large error associated low concentration of glucose in the blood and thus a small change in the refractive index of the corresponding phase shift in comparison with the contribution of blood plasma. In addition, the effects of dynamic scattering on shaped formations of blood will result in considerably the output fluctuation of the refractive index and the noise interference signal.

There is a method of determining the level of glucose in the blood based test strips reagent (see application for a patent of the Russian Federation No. 97105194, IPC G01N 33/66, G01N 33/52). The test strip with the reagent for use in a device for determining the concentration of glucose in a sample of whole blood containing optical means of detecting the intensity of light at wavelengths of about 635 nm and about 700 nm reflected from at least part of the matrix is located near one of the edges of the strips, characterized in that the matrix contains (a) a receiving surface of the sample for receiving a sample of whole blood and the passage portion in the direction of the surface of the testing, opposite, surface test with a reflection coefficient at a wavelength of about 700 nm, that is, when the surface testing becomes wet, there is a change, which is essentially equivalent to such change caused by the absorption of hemoglobin in the blood, (b) a structure that selectively retards the passage of erythrocytes through the matrix and minimizes the destruction of cells in the matrix, making any part of the sample, which is visible from the surface of the test does not absorb light in any appreciable degree at a wavelength of about 700 nm, and (C) a reagent for indicating the concentration of glucose by creating on the surface of the test changed what I reflection coefficient at a wavelength of about 635 nm.

However, this method does not allow to evaluate the content of glucose in the blood cells.

The closest is a non-invasive method of determining the concentration of glucose in human blood (see RF patent №2295915, IPC AV 5/1455). The method is carried out by irradiation with a laser beam in the zone of maximum accumulation of the blood vessels in the mucous membrane, reception and conversion apparatus, by identifying the orientation of the polarization vector and the intensity of the reflected radiation, and calculation of the concentration of substances in the blood. For irradiation using a laser beam with zero polarization and wavelength in the range of 0.5 μm and 2.1 μm. Pre-configure the analyzer Polaroid on the point of the local absorption of laser radiation in the mucous tissue of the detected substances, fix change the orientation of the polarization vector of the reflected radiation at the points of local absorption of laser radiation by the angle of its rotation, and is judged on the concentration of the substance in the magnitude of the rotation angle of the polarization vector. The use of the invention improves the accuracy of measurement and to expand the number of detected substances.

However, this method has low accuracy, since the blood vessels, for example, in the dermis are at a depth of about one millimeter from the stratum corneum. When distributing linearly polarisavenue what about the light in the surface layers of the skin, which are anisotropic, strongly varying spatial areas of sensing, should lead to ambiguous interpretation of the measurement results in the presence or absence of blood, not to mention the influence of blood glucose concentration is three orders of magnitude smaller than, for example, hemoglobin.

The aim of the invention is the ability to determine the glucose is not only in plasma but also non-invasive measurement of glucose within the selected live any blood cells, including erythrocytes, lymphocytes, monocytes, platelets and other cell-shaped formation of blood. This method should allow to determine the effectiveness of the penetration of glucose through the plasma membrane of living cells under the action of, for example, the hormone insulin or various drugs and to investigate the dynamics of change of glucose in normal and abnormal blood cells.

Method for determination of glucose in plasma and blood cells, including contact sampling of arterial whole blood of the patient, non-invasive sensing of the blood sample in the cuvette optical radiation of visible or near IR range, the measurement of the intensity of the reflected backward optical radiation, according to the decision as an optical probe radiation using a laser beam which is spatially focus the display in the selected blood cell (erythrocyte, lymphocyte, platelet or blood plasma, the wavelength of the laser radiation is chosen in the range 570-1100 nm, not mentioned in the spectral absorption band of hemoglobin and water, measured the Raman spectrum in the spectral range from Stokes frequency shift of 100 cm-1to 3200 cm-1from the internal fixed volume of blood cells or blood plasma, determined by confocal volume defined by the diameter of paired microderm, pre-measure the Raman spectrum of glucose, hemoglobin and water concentrations, typical content in plasma and within the erythrocyte, choose Stokes spectral components in the spectrum of Raman scattering, the corresponding characteristic resonant vibrations of molecules of glucose per minimum Stokes spectral component of the Raman scattering of hemoglobin and water, measure the amplitude of the Stokes spectral component of glucose and compared with the amplitudes of Stokes spectral component of the calibration curve Raman scattering glucose obtained with known concentrations of glucose solution, determine the concentration of glucose in plasma or separate the blood cells.

For analysis using microprobe whole blood with no more than 10 ál.

The main advantage of p is izlagaemogo non-invasive method for determination of glucose inside the erythrocyte and lymphocyte or platelet whole blood it is possible to study the dynamics of the penetration of glucose across plasma membrane cell under the action of insulin or other hormones or drugs.

The invention is illustrated by drawings.

Figure 1 presents the block diagram of the device for implementing the proposed method for the determination of glucose in red blood cells of whole blood.

Positions on the drawings indicated:

1 - cell (erythrocyte) in the plasma of whole blood;

2 - cover the glass on the X-Y preparationtime;

3 - a micro with a focal length that is tunable by the depth of the sample;

4 - separating mirror;

5, 9 - paired microdamage;

6 - narrowband resonant optical filter that absorbs only the laser light;

7, 10 - focusing lens;

8 is a reflective diffraction grating;

11 is a CCD matrix or line of photodetectors;

12 is a personal computer;

13 is a laser with a specific wavelength, causing Raman scattering in plasma or blood cells.

Figure 2 presents the Raman spectrum (CU) glucose in saline solution with a concentration of 1 μg/ml, typical of glucose in the blood plasma of a person, measured using the setup shown in figure 1 when using He-Ne laser with a wavelength of 633 nm. The scale on the x-axis (frequency shift)·103cm-1on the y-axis of the relative intensity of the CR.

3 shows the Raman spectrum of races is eania hemoglobin concentration of 130 μg/ml typical content hemoglobin in red blood cells, measured using the setup shown in figure 1, when using He-Ne laser with a wavelength of 633 nm. The scale on the x-axis (frequency shift) of 103cm-1on the y-axis of the relative intensity of the CR.

Figure 4 presents a range of local Raman scattering of individual erythrocytes from droplets (10 μl) of whole capillary blood, measured using the setup shown in figure 1 when using He-Ne laser with a wavelength of 633 nm in the mode of non-invasive probing (probing laser power W=2 mW, the measurement time CR spectrum τ=0.1 sec); the Scale on the x-axis (frequency shift)·103 cm-1on the y-axis of the relative intensity of the CR.

The method is as follows.

As exciting Raman scattering in the plasma of whole blood 1 and shaped formations (erythrocytes, leukocytes) uses a laser 13 with a wavelength in the range 570-1100 nm, not falling within the absorption band of the chromophores cells (hemoglobin) and water. Laser radiation passed through the focusing lens 10, microdamage 9 using tunable micro 3 focuses on some selected depth of the erythrocyte or leukocyte or in the blood plasma in a drop of whole blood on top of the glass, before artelino treated with anticoagulant (Trilon B, heparin), and cross-sectional configuration is performed using the X-Y preparatively 2. The use of two paired microderm 5, 9 and dividing mirror 4 is designed to measure the back-reflected optical radiation from the diffraction microvolume, a smaller volume of blood cells and determined by the diameter of the paired apertures. Using narrow-band resonant optical filter 6 from reflected back to the optical radiation is selectively absorbed by the narrow-band resonant filter only laser radiation, and scattered by the molecules and shifted in frequency relative to the laser by an amount determined by the characteristic vibration frequencies of molecules and past narrowband resonant optical filter 6, in the form of a parallel optical beam formed by the focusing lens 10, falls on the reflective diffraction grating 8, which is a definite transformation of the frequency spectrum in the corner, and the measurement of the Raman scattering spectrum is carried out using a CCD sensor or a line of photodetectors 11 with integrated ADC and the personal computer 12. Using the proposed method previously performed calibration, including the measurement of Raman scattering spectrum of glucose in saline solution with a concentration typical of plasma (1 is g/ml) (Figure 2), measurement of hemoglobin with a typical concentration of its content in erythrocytes (130 mg/ml) (Figure 3) and similarly Raman spectrum for saline. In the Raman spectra of finding the spectral components corresponding to the maximum spectral component of glucose in the frequency range corresponding to the minimum of the spectral component of hemoglobin and water (for example, in the spectral range of 1000-1200 cm-1) and the amplitude of the spectral component CR of glucose in the blood plasma or blood cells determine its concentration by comparing the amplitude of spectral components with CU glucose in saline solution with known concentration.

In conclusion, it should be noted potential proven method. It is known that the average volume of an erythrocyte is 80-95 μm3with hemoglobin 25-34 PG, and analyzed confocal volume sensing, for example, at a wavelength of a visible range is 0.5 μm3therefore, it becomes possible to achieve the degree of intracellular locality more than two orders of magnitude.

Figure 5 presents a range of local Raman scattering of individual leukocyte of droplets (10 μl) of whole capillary blood, measured using the setup shown in figure 1 when using He-Ne laser with a wavelength of 633 nm proregime non-invasive probing (probing laser power W=2 mW, the measurement time of the KR spectrum τ=0.1 sec); the scale on the x-axis (frequency shift) of 103cm-1on the y-axis of the relative intensity of the CU. Unlike erythrocyte CU range of individual leukocyte does not contain the characteristic resonance peaks caused by the molecules of hemoglobin, but there are spectral components of the water molecules. In the spectral range of 500-1500 cm-1there is the minimum amplitude of the spectral component from a leukocyte. Therefore, in this region of the spectrum, you can find the resonant peaks associated with glucose molecules. In contrast to known methods for the determination of glucose in blood, the inventive method allows you to explore the content of glucose in an individual blood cell and, potentially, to assess the dynamics of diffusion of glucose through the membrane of living cells under the influence of hormones (insulin or glucagon), which is very important for the development of effective treatments for diabetes.

Method for determination of glucose in the blood cell, including contact sampling of arterial whole blood of the patient, non-invasive sensing of the blood sample in the cuvette optical radiation of visible or near IR range, the measurement of the intensity of the reflected backward optical radiation, characterized in that the optical probe radiation using a laser beam, which is output spatial focus in individual blood cell, selected from erythrocytes or leukocytes, and the wavelength of the laser radiation is chosen in the range 570-1100 nm, not mentioned in the spectral absorption band of hemoglobin and water, measured the Raman spectrum in the spectral range from Stokes frequency shift of 100 cm-1to 3200 cm-1from a fixed volume inside the blood cells, determined by confocal volume defined by the diameter of paired microderm, pre-measure the Raman spectrum of glucose, hemoglobin and water concentrations, typical content inside blood cells, choose Stokes spectral components in the spectrum of Raman scattering, the corresponding characteristic resonant vibrations of molecules of glucose per minimum Stokes spectral component of the Raman scattering of hemoglobin and water, measure the amplitude of the Stokes spectral component of glucose and compared with the amplitudes of Stokes spectral component of the calibration curve Raman scattering glucose obtained with known concentrations of glucose solution, determine the concentration of glucose in a single blood cell.



 

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