RU2722898C1 - Method for detecting pathological changes in structural elements of erythrocyte membranes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to hematology, and can be used for detecting pathological changes in structural elements of erythrocyte membranes (EM). For this purpose, using an atomic power microscope, an image of the EM surface is obtained and decomposed into images of surfaces of three scales P₁, P₂, P₃. Scale P₁ corresponds to flickering parameters EM. Scale P₂ corresponds to the parameters of the spectrin matrix EM. Scale P₃ corresponds to parameters of protein clusters EM. Heights of detected topological nanostructures, their average values and their mean square deviations are measured. Then probability of deviation of height values of nanostructures P₁, P₂, P₃ from normal values in the control is determined. At the same time deviation of value P₁ more than 30 % for P₁ indicates deviation of value of flickering amplitude. Value P₂ more than 30 % for P₂ testifies to deviation of physical characteristics of spectrinum matrix. P₃ value more than 30 % for P₃ indicates clustering of intermembrane proteins.

EFFECT: invention makes it possible to quantify the degree of the pathological factor effect on the erythrocyte membrane state.

1 cl, 2 tbl, 11 dwg

Description

The invention relates to medicine, namely to the biophysics of human cell membranes, to hematology and can be used to study the membranes of red blood cells (ME) and the diagnosis of various pathological processes in the blood.

State of the art

A number of methods are known for studying the structural components of MEs using various biophysical and physicochemical methods. Basically, the known methods relate to the research of each of the membrane elements separately: the amplitudes of the self-oscillations of the membrane (flickering), spectrin matrix, protein clusters.

A number of methods are known for assessing the state of ME by the effect of damage to the membranes. In particular, the ME mechanical fragility test is known (ERYTHROCYTE MECHANICAL FRAGILITY TEST US 2014017718). In accordance with this method, a mechanical effect on the cells is performed and the level of hemolysis is evaluated. However, the integral effect of damage to all of the above mentioned membrane components is evaluated.

It should be noted that all known methods that evaluate the mechanical fragility of membranes, as well as the above, are based on a violation of the membrane structure and subsequent hemolysis of the cells. This means a violation of the integrity of the ME in the diagnostic process. In this case, there is a violation of the spectrin matrix, flickering, protein complexes and cells in general.

At the same time, methods for studying membranes are known in which membranes and cells are not destructively affected.

A known method for controlling the speed of ME fluctuations using a digital holographic microscope (Dynamic parameters based red blood cell membrane fluctuation inspection method and digital holographic microscopy used in the same KR 101805152). In addition, a number of scientific articles are devoted to studying the process of ME fluctuations (flickering): Madalena Costa, Ionita Ghiran, S.-K. Peng, Anne Nicholson-Weller and Ary L. Goldberger "Complex dynamics of human red blood cell flickering: Alterations with in vivo aging" // Phys Rev E Stat Nonlin Soft Matter Phys. 2008 August; 78 (2 Pt 1): 020901).

A known method for studying changes in the spectrin matrix as an indicator of cellular pathology (Method for detecting cellular pathology WO 9002949). However, the immunochemical method used to analyze the decay products of spectrin, a component of the cytoskeleton, is not possible to measure the spatial dimensions of the studied spectrin matrix.

A known method for determining compacted protein-lipid regions in MEs of varying degrees of maturity using the Bio Vision computer program (RU 2390020) involves a combination of optical and computer methods for visualizing intermembrane objects. However, the resolution of the method remains insufficient to visualize nano-objects of blood cell membranes.

Closest to the claimed method according to the set of essential features is a method for integrated assessment of the strength of the protein-lipid interaction in ME, in accordance with which the fluorescence parameters are measured, which reflect the amount and degree of immersion of membrane proteins in lipids (RU 2187112). Adopted as a prototype. However, the fluorescence method, whose resolution limit is 500–1000 nm, does not allow obtaining images and studying processes in the studied objects at distances shorter than several light wavelengths, which significantly exceeds the size of membrane nanostructures.

The claimed invention is aimed at solving the problem of identifying pathological changes in the structural elements of ME.

Use in laboratory and clinical practice of the proposed method allows to achieve several technical results:

• approximation of the studied object (ME and its structure) to the native conditions of existence by maintaining the integrity of the cell and the cell membrane during the study;

• simultaneous comprehensive measurement of the characteristics of the main nanostructures of MEs (flickering elements, spectrin matrix, protein clusters);

• simultaneous assessment of the probability of deviation of the characteristics of all three of the above nanostructures from the control values;

• improving the accuracy of the study of ME nanostructures due to the high resolution of the obtained images using the atomic force microscopy (AFM) method,

• the ability to quantify the degree of influence of a pathological factor on the state of the blood.

These technical results in the implementation of the invention are achieved due to the fact that, as in the known method, use the physical method of obtaining information about the state of proteins and lipids in the membrane without violating its integrity.

A feature of the proposed method is that the following stages are carried out: they scan the surface of P cells using an AFM in a field of size (1.2 ÷ 1.6) × (1.2 ÷ 1.6) μm ² , decompose the resulting image of surface P on images of surfaces of three spatial scales P ₁ P ₂ , P ₃ corresponding to the parameters of physiological structures of ME: scale P ₁ for which the typical spatial period of the structures of this surface L ₁ = 400 ÷ 900 nm corresponds to the flickering parameters of ME, scale P ₂ for which typical the spatial period of structures of a given surface L ₂ = 50–200 nm corresponds to the parameters of the spectrin matrix of ME, scale P ₃ for which the typical spatial period of the structures of this surface of L ₃ = 5–40 nm corresponds to the parameters of protein clusters of ME. The heights h ₁ , h ₂ , h _{3 of the} identified topological nanostructures on the selected three surfaces are measured; calculate the average values of h _1cp , h _2cp , h _3cp and the mean square deviations of σ ₁ , σ ₂ , σ _{3 of} these heights, determine the probability of deviation of the heights of the nanostructures P ₁ , P ₂ , P ₃ for all three surfaces from the values corresponding to physiological parameters ME structures in a normal control smear. The value of P ₁ more than 30% for the surface of the first scale P ₁ indicates the deviation of the magnitude of the flickering amplitude from normal values, the value of P ₂ more than 30% for the surface of the second scale P ₂ indicates the deviation of the physical characteristics of the spectrin matrix from normal values, the value of P ₃ more than 30 % for the surface of the third scale P ₃ indicates the occurrence of clustering of intermembrane proteins.

Disclosure of the invention

With inflammatory processes in the body, with infection of the body, with endogenous and exogenous intoxication, with sepsis, with the action of ionizing radiation, with prolonged storage of donated blood, with transfusion, etc., pathological processes develop in the blood - oxidative, immune, etc. As a result of this the functioning of blood cells, in particular red cells, is impaired, which may result in tissue hypoxia and necrosis. There may also be changes in the rheological properties of the blood, disrupting the blood supply to the tissues. With the development of pathological processes in the blood, the morphology of red blood cells changes - the shape of the cells and / or their fusion, which indicates a violation of the stable state of ME. The trigger mechanism for changing the morphology of red blood cells is a violation of nanostructures in ME.

The origin and development of pathological processes at the molecular level in ME is an indicator of the pathological effect of physical and chemical factors on the blood.

The following structural elements can be attributed to ME nanostructures: flickering elements, spectrin matrix, protein clusters.

Various factors can have a pathological effect on various studied structures.

It has been experimentally shown that membrane fluctuations (surface P ₁ ) change depending on the conditions for the existence of red blood cells, in particular, there is a difference for young and old cells, changes are observed during the introduction of malarial plasmodium into red blood cells, with sickle cell anemia. It is also suggested that measuring flickering characteristics may be useful for evaluating cellular processes and for diagnosing diseases (C Monzel and Co. Sengupta Measuring shape fluctuations in biological Membranes .: 2016 J. Phys. D: Appl. Phys. 49 243002).

The main function of the spectrin matrix (P ₂ surface) is to maintain the stability of the cytoskeleton of the membrane and its mechanical properties. The clinical manifestations of the disturbance of the elements of the spectrin matrix are hemolytic anemia, spherocytosis, elliptocytosis (Zhang Ruil, Zhang ChenYul, Zhao Qi & Li DongHail Spectrin: Structure, function and disease // Sci China Life Sci December (2013) Vol. 56 No. 12: 1076 -1085).

It was shown that during prolonged storage of donated blood in special solutions at 4 ° C in ME during the first two weeks of storage, band 3 proteins cluster in large aggregates (P ₃ surface), which is one of the reasons for vesiculation (Brad S. Karon, James D. Hoyer, James R. Stubbs, and David D. Thomas. Changes in Band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells // Transfusion. 2009 July; 49 (7): 1435-144) .

The need for a comprehensive determination of the state of the above mentioned membrane elements is due to the fact that the ability of red blood cells to deform in vessels with a diameter smaller than the cell size is determined by the state of all three of these structures at the same time. In addition, the characteristics of these elements are structurally and functionally interconnected. Measuring them separately at different points in time will lead to a distortion of cause-effect relationships in the analysis of processes in the cell and in the membrane.

The method is as follows.

Preliminary measure the corresponding control (normal) parameter values for ME 30 healthy donors.

To approach the native conditions, the morphology of erythrocytes is preserved when preparing smears of a monolayer of cells using a special V-sample device (Austria). Using AFM scanning, cell images are first obtained in a field of 50–100 μm. Then, an AFM image of the selected individual cell is obtained. An AFM image of the surface P of the membrane section is obtained (1.2 ÷ 1.6) × (1.2 ÷ 1.6) μm ² . The choice of the size of the membrane surface area is determined by the sizes of the studied surface elements - flickering elements, spectrin matrix and membrane clusters.

Using the AFM software, the obtained image of the surface P is decomposed into three surface images of three spatial scales P ₁ , P ₂ , P ₃ , and P-P ₁ + P ₂ + P ₃ . These three images correspond to the parameters of the physiological structures of the ME: the scale P ₁ for which the typical spatial period of the structures of a given surface L ₁ = 400 ÷ 900 nm corresponds to the flickering parameters of the ME, the scale P ₂ for which the typical spatial period of the structures of this surface L ₂ = 50 ÷ 200 nm corresponds to the parameters of the ME spectrin matrix, P ₃ scale for which the typical spatial period of structures of a given surface L ₃ = 5–40 nm corresponds to the parameters of ME protein clusters.

To obtain an image of a surface of the first scale P _{1, the} image of the initial surface P is smoothed by a filter using AFM software (a mask of 9 points), then the image P _{1 is} subtracted from the image P and an image P ₂ + P _{3 is} obtained, it is smoothed by a filter (3 points mask) and get the image of the second scale P ₁ , from the image P ₂ + P ₃ subtract the image P ₂ and get the image of the surface of the third scale P ₃ .

For each of the three surface images, the heights h ₁ , h ₂ , h _{3 of the} identified topological nanostructures are measured. Then calculate the average values of h _1cp , h _2cp , h _3cp and the standard deviations σ ₁ , σ ₂ , σ _{3 of} these heights.

For each of the three surface images, the probability P ₁ , P ₂ , P _{3 of} the fact that the heights in the test sample are beyond the control values of the corresponding surfaces of the first, second, third scales is calculated.

The probability value indicates the fact that the characteristics of the membrane nanosurface deviate from normal values and allows us to quantitatively evaluate the degree of influence on the examined structure i:

• the value of P ₁ more than 30% for the surface of the first scale P ₁ indicates the deviation of the flickering amplitude from normal values,

• the value of P ₂ more than 30% for the surface of the second scale P ₂ indicates a deviation of the physical characteristics of the spectrin matrix from normal values,

• P ₃ value _of more than 30% for the surface of the third P ₃ scale indicates the occurrence of clustering of intermembrane proteins.

With a P _i value of more than 60%, the effect on structure i is considered strong, with a P _i value of 30% to 60%, the effect is considered moderate (moderate), with a P _i value of 5% to 30%, the effect is considered weak, with a P _i value of less 5% believe that this factor did not show structural changes i.

Examples of carrying out the invention

Measurement of heights h ₁ , h ₂ , h _{3 of} topological nanodefects of surfaces of three scales P ₁ , P ₂ , P ₃ ME of the blood of a healthy person (control sample) in vitro.

A smear of a monolayer of cells from the blood of a healthy donor was prepared on a glass slide. Then, the obtained smear was scanned using an NTEGRA PRIMA AFM (NT-MDT, Zelenograd, RF). For scanning, standard NSG01 cantilevers (5 n / m) were used. For scanning, fields of 100 × 100 μm ² and 10 × 10 μm ² were selected. The number of scan points along one line was 512 and 1024, respectively.

On the obtained image of the cell surface, a fragment with an area of 1.2-1.5 × 1.2-1.5 μm was isolated. This surface was designated P. The image of the surface of the ME from the blood of a healthy donor and the profile of the heights of the topology of the nanosurface of the ME in a given section indicated by a dotted line are shown in FIG. 1.

The smoothing technique was applied to the image of the surface P using 9 × 9 filters using AFM software. Thus, a surface image of the first scale P ₁ was obtained (shown in Fig. 2a). A typical profile of the heights of the topology of a nanosurface in a given cross section after smoothing using 9 × 9 filters is shown in FIG. 2b.

The characteristic parameters of the profile of the topological structure of the erythrocyte nanosurface of the first scale P ₁ are the height h ₁ and the spatial period L ₁ . The value of L ₁ is the typical spatial period of the structures and the given surface of the first scale, that is, the distance between the troughs or peaks on the profile (Fig. 2b.) Empirically it has been established that normally these values are h _1norm = 4.5 ± 1 nm and L _1norm = 700 ± 150 nm.

Next, the image of the surface P _{1 was} subtracted from the image of the initial surface P and the surface P ₂ + P _{3 was} obtained. Smoothing technique using 3 × 3 filters using AFM software was applied to the obtained image P ₂ + P ₃ . Thus, a surface image of the second scale P ₂ was obtained (shown in Fig. 3a). A typical profile of the heights of the topology of the nanosurface of the second scale P ₂ in a given section is shown in FIG. 3b.

The characteristic parameters of the profile of the topological structure of the erythrocyte nanosurface of the second scale P ₂ are the height h ₂ and the spatial period L ₂ . Empirically, it was found that normally these values are h _2norm = 1.6 ± 0.7 nm and _L2norm = 120 ± 80 nm.

Next, from the previously obtained image P ₂ + P ₃ subtracted the image of the surface P ₂ . As a result, a surface image of the third scale P ₃ (shown in Fig. 4a) and the corresponding height profile (presented in Fig. 4b) were obtained.

The characteristic parameters of the profile of the topological structure of the erythrocyte nanosurface of the third scale P ₃ are the height h ₃ and the spatial period L ₃ . Empirically, it was found that normally these values are h _3norm = 0.7 ± 0.2 nm and L _3norm 40 ± 10 nm.

2. Change in the ME nanosurface with the addition of a fixative - glutaraldehyde in the blood (in vitro)

Glutaraldehyde is widely used to fix cells during their research. However, the action of the fixative leads to changes in the structure of cell membranes. This must be taken into account when studying the topological nanostructure of blood cell membranes after exposure to blood of various physicochemical factors.

To study the effect of glutaraldehyde on the nanostructure of the surface of erythrocyte membranes, the authors conducted model experiments in which 50 μl of 1% glutaraldehyde was added to 50 μl of red blood cells extracted from donor blood. After 10 min of incubation, the red blood cells were washed three times in a 0.9% NaCl solution using a centrifuge (RPM 3000 min ^-1 ) and a cell monolayer was formed. AFM images of the nanosurface of the ME were obtained on three scales and the corresponding heights h _1glut , h _2glut , and h _{3glut were measured} . Table 1 shows the values of the parameters h _i = h _{i averag} ± σ _i for the heights of surfaces of different scales П _i in the control sample and in the sample after the action of glutaraldehyde, i = 1, 2, 3. The presented results indicate a strong effect of glutaraldehyde on the amplitude flickering, as well as on protein clusters. There is practically no effect of glutaraldehyde on the spectrin matrix.

In FIG. Figure 5 shows the distribution functions of the heights of topological nanostructures h ₁ on the first-order surface P ₁ for membranes of control red blood cells and after the action of glutaraldehyde. The proportion of h ₁ coming out of the control values for the surface of the first scale P ₁ , in this case is 80%.

In FIG. Figure 6 shows the distribution functions of the heights of topological nanostructures h ₂ on the second-order surface P ₂ for the membranes of control erythrocytes before and after the action of glutaraldehyde. The proportion of h ₂ that leaves the control values for the surface of the second scale is in this case about 0%.

In FIG. Figure 7 shows the distribution functions of the heights of topological nanostructures h ₃ on the third-order surface P ₃ for membranes of control erythrocytes before and after the action of glutaraldehyde. The proportion of h ₃ coming out of the control values for the surface of the third scale is in this case 100%.

3. Change in the nanosurface of ME under the action of hemin on red blood cells in vitro

Gemin is a substance formed by the action of hydrochloric acid on hemoglobin. With a stomach ulcer, blood can enter the gastric juice as a result of bleeding. Under the influence of hydrochloric acid of gastric juice, hemoglobin of blood gradually turns into hydrochloric hemin. When hemoglobin is oxidized, ferrous iron is converted to ferric. The presence of hemin in the blood can cause pathological effects.

The authors conducted model experiments in which hemin solution was added to the blood in vitro. To prepare a solution, 50 mg of hemin powder was dissolved in 1 ml of 0.5 M NaOH in distilled water. Then, 5 ml of distilled water was added to 1 part of this stock solution. 35 μl of this hemin solution was added to microvets containing 140 μl whole blood. The final concentration of hemin in the blood was 2 mm. The incubation time of hemin in the blood was 5 minutes.

On the ME surface, the formation of domains with a granular structure was observed (shown in Fig. 8 a). The surface profile in a given section is shown in Fig. 8b.

To quantify the size of topological nanodefects, the initial surface P was decomposed on a surface of three scales (shown in Figs. 9a, 10a, 11a). For AFM images of each surface, the profiles of the heights of the nanosurface in given cross sections indicated by dashed lines are shown (Fig. 9b, 10b, 11b).

Table 2. shows the average values and standard deviations of the parameters h _i for the cell membranes in the control sample and in the test after the action of hemin.

A significant change in the average height h _{1 of the} nanosurface of the first scale was observed. The presented results indicate a strong effect of hemin on the amplitude of flickering of the membrane.

The average height of the nanosurface of the second scale has substantially changed. This indicates a strong violation of the spectrin matrix when hemin acts on the blood. In this case, the height of the nanosurface of the third scale is practically unchanged. This indicates that there is practically no effect of hemin on protein clustering.

Thus, the authors of the proposed method identified a universal measurable set of parameters at the same time, indicating the presence of a disease, physiological change, reaction to treatment, reaction to physico-chemical effects (for example, to ionizing radiation).

Claims (1)

A method for detecting pathological changes in the structural elements of erythrocyte membranes (MEs), including visualizing the surface of MEs in a cell monolayer, characterized in that the following steps are carried out: the surface of P cells is scanned using an atomic force microscope in a field of 1.2 ÷ 1.6 × 1 in size , 2 ÷ 1.6 μm ² , the obtained image of the surface P is decomposed into images of surfaces of three spatial scales P ₁ , P ₂ , P ₃ corresponding to the parameters of the physiological structures of the ME: scale P ₁ for which the typical spatial period of the structures of this surface is L ₁ = 400 ÷ 900 nm corresponds to the ME flickering parameters, scale П ₂ , for which the typical spatial period of the structures of a given surface L ₂ = 50 ÷ 200 nm corresponds to the ME spectral matrix parameters, scale П ₃ , for which the typical spatial period of the structures of a given surface L ₃ = 5–40 nm corresponds to the parameters of ME protein clusters; measure the heights h ₁ , h ₂ , h ₃ identified topological nanostructures on the selected three surfaces; calculate the average values of h _icp , h _2cp , h _3cp and the standard deviations σ ₁ , σ ₂ , σ _{3 of} these heights; determine the probability of deviation of the heights of the nanostructures P ₁ , P ₂ , P ₃ for all three surfaces from the values corresponding to the parameters of the physiological structures of ME in a normal control smear; the value of P ₁ more than 30% for the surface of the first scale P ₁ indicates a deviation of the magnitude of the flickering amplitude from normal values, the value of P ₂ more than 30% for the surface of the second scale P ₂ indicates a deviation of the physical characteristics of the spectrin matrix from normal values, the value of P ₃ more than 30 % for the surface of the third scale P ₃ indicates the occurrence of clustering of intermembrane proteins.

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