RU2433175C2 - Method of cell analysis by means of biochip - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: described is method of cell analysis by means of biochip, containing immobilised molecules of substances, able of binding with molecules, which are on the surface of cells, includes incubation of biochip with suspension of cells. Then, washing of biochip from nonspecifically bound cells is carried out. After that, fixation and staining of cells, reading of results and estimation of quantity of cells, which bound in one or several parts of biochip of the given area, are performed. In conclusion, analysis of the image of bound cells is carried out. Before carrying out fixation from biochip surface excess of liquid is removed, without permitting it to dry.

EFFECT: improvement of technology.

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Description

The invention relates to medicine, but can also be used in veterinary medicine and biology.

A known method for the study of cells using immunological biochips (see Liu AY Differential Expression of Cell Surface Molecules in Prostate Cancer Cells // Cancer Research. 2000, Vol.60, p. 3429-3434), which consists in the fact that the biochip containing immobilized antibodies are incubated with a suspension of cells. Cells having corresponding antigens on their surface bind to antibodies immobilized in strictly defined regions of the substrate. Then the biochip is washed from cells that did not bind to immobilized antibodies. Reading the result is carried out by microscopic examination of the biochip, as a result of which it is determined in which particular areas of the cell binding occurred. Thus, the presence of detectable surface antigens in the studied cells is established.

The disadvantage of this method is that it is not provided for the possibility of staining and morphological studies of cells. As a result, in the study of suspensions containing several types of cells, it is impossible to determine exactly which cells one or another of the determined antigens is present.

A known method for the study of cells using immunological biochips (see A.V. Shishkin, I.I. Shmyrev, S.A. Kuznetsova, N.G. Ovchinina, A.A. Butylin, F.I. Ataullakhanov, A.I. Vorobyov “Immunological biochips for the parallel determination of surface antigens and morphological study of cells”, Biological membranes, 2008, Volume 25, No. 4, pp. 277-284), taken as a prototype, namely that the biochip containing immobilized antibodies is incubated with cell suspension. Cells with corresponding antigens on their surface bind to antibodies immobilized in strictly defined areas of the substrate (biochip spots). Then, the biochip is washed from cells that did not specifically bind to the surface of the biochip, but did not bind to immobilized antibodies. As a result, only cells bound to antibodies remain on the biochip surface. After that, the biochip is dried in air, fixed with methanol, stained according to Romanovsky-Giemsa and a morphological study of the bound cells is carried out. The result is read in which an image of the biochip spots (areas of the biochip surface with immobilized antibodies) in which the cells bind is obtained. The density of cell binding in each spot of the biochip is determined (the ratio of the number of connected cells to the surface area of the site). Perform an analysis of the result. In this case, based on the values of the cell binding density obtained for each spot, the content in the suspension of cells having the corresponding surface antigens is calculated. In addition, an image analysis of cells (morphological examination) is carried out by direct microscopic examination of cells bound in the area of the biochip stains or by studying images obtained by microphotography of stains. A morphological study allows us to determine which types of cells have a particular antigen on their surface.

The disadvantage of this method is that in the process of drying the biochip there is a significant change in the morphological characteristics of the cells. A morphological study of cells shows their deformation, many cells have signs of apoptosis. Some cells are destroyed. This makes their recognition difficult. In spots with a high density of cell binding, in some cases the cells look “merged” with each other. This makes it difficult to determine the density of their binding. The reason is that when the biochip is dried, water evaporates from the droplets of the buffer solution remaining on its surface. This leads to a gradual increase in salt concentration. A change in osmotic pressure leads to cell deformation and, in all likelihood, causes processes leading to the initiation of apoptosis mechanisms.

The objective of the claimed invention is to preserve the morphological characteristics of cells bound to the biochip, to increase the accuracy and reliability of the results obtained by eliminating possible errors during morphological studies and determining the density of cell binding.

The problem is solved due to the fact that according to the method of studying cells using a biochip containing immobilized molecules of substances capable of binding to molecules located on the surface of cells, including incubating the biochip with a suspension of cells, washing the biochip from non-specifically bound cells, fixing and staining cells , reading the result, estimating the number of cells bound in one or more parts of the biochip of a given area, analyzing the image of the bound cells, before conducting phi sation from the surface of the biochip remove excess fluid, while avoiding its drying.

A long-term preparation for microscopic studies is prepared from a biochip.

During the removal of excess washing liquid, the biochip is placed in an atmosphere with high humidity.

The result of using the invention is to increase the accuracy and reliability of the results due to the preservation of the morphological characteristics of the cells bound to the biochip and the elimination of possible errors during their morphological studies and determination of cell binding density.

Using the inventive method, it is possible to prevent a change in the shape of the cells, the shape of the nucleus and other morphological characteristics of the cells caused by a change in the osmotic pressure during evaporation of water from the droplets of the buffer solution remaining on the surface of the biochip and apoptotic processes in the cells. When fixing fluid is added immediately after eliminating the excess washing solution, cell death occurs without triggering apoptosis. However, there are also no changes due to an increase in the concentration of salts.

After staining, a long-term preparation for microscopic examination can be prepared from the biochip. This allows you to protect its surface with the bound cells from damage and repeatedly microscopic examination using immersion lenses. In addition, this allows the biochip to be stored indefinitely after the study.

Performing the manipulations associated with removing excess washing solution in conditions of high humidity allows you to slow down or prevent the drying of the biochip surface and allows you to increase the allowable time interval between removing the excess washing solution and placing the biochip in the fixing liquid.

The claimed method is illustrated by drawings (figures 1-4).

Figure 1 presents the results of a study using a biochip of cells of patient C., 51 years old, with a diagnosis of chronic B-cell lymphocytic leukemia (B-CLL). The diagram below shows the percentage values of cell binding density in test sections of the biochip with immobilized antibodies. These values numerically correspond to the percentage in the test sample of cells having each of the determined antigens.

Figure 2 presents micrographs of the spots (with immobilized antibodies specific for the CD23 antigen) of two biochips, on which the study of lymphocytes isolated from the peripheral blood of a patient with a diagnosis of chronic B-cell lymphocytic leukemia was performed. Coloring according to Romanovsky-Giemsa. 1000x magnification. a) The study was conducted according to the claimed method. The cell structure is well preserved. b) The study was carried out by the method taken as a prototype. The cells are deformed, fragmentation of the nucleus is noted in some of the cells. Some cells are destroyed.

Figure 3 presents the results of determination using a biochip of the percentage of cells having various antigens. Cells of patient B. 77 years. Diagnosis: hairy cell leukemia (ON). The diagram below shows the percentage values of cell binding density in test sections of the biochip with immobilized antibodies. These values numerically correspond to the percentage in the test sample of cells having each of the determined antigens.

Figure 4 presents a micrograph of the spot (with immobilized anti-CD19 antibodies) of the biochip, on which a study was conducted of lymphocytes isolated from the peripheral blood of a patient with a diagnosis of hairy cell leukemia. The study was conducted according to the claimed method. The cell structure is well preserved. The outgrowths of the cytoplasm resembling villi are clearly visible, which is characteristic of the cells of this tumor. 1000x magnification. Pappenheim stain.

The claimed method is as follows. The biochip, which is a transparent solid substrate with antibodies or other molecules immobilized in predetermined areas (spots), capable of binding to surface cell molecules, is fixed in a cuvette, Petri dish, or other container. The test cell suspension is poured into the container and incubated. Cells settle on the surface of the biochip and come into contact with immobilized antibodies. If the cells have corresponding surface antigens, they bind in these areas (spots) of the biochip surface. Then, the biochip is washed with an isotonic solution to eliminate cells that are not bound to antibodies. After washing, only those cells that have the corresponding surface antigens remain bound in the area of the biochip spots. Immediately after washing, the remaining washing solution is drained, and its drops are removed from the walls of the tank with a pipette, without touching the biochip. After that, a fixing liquid, for example methanol, an alcohol-formalin solution or another liquid, is poured into a container with a biochip and incubated for the required time. The time interval between removal of excess washing solution and the addition of fixing fluid should not exceed 15-30 seconds. But it can be increased if this stage of the analysis is performed at high air humidity, which prevents the evaporation of water from the surface of the biochip.

After processing the biochip with a fixing liquid (12-15 minutes for methanol, 30 seconds for an alcohol-formalin solution), it is drained. At this stage, drying the biochip is allowed. A staining solution is poured into the cuvette, for example, a solution of azure II and eosin during Romanovsky-Giemsa staining. It is possible to stain the drug in any other known manner. In particular, it is possible to carry out various variants of cytochemical staining, which makes it possible to detect the presence of various substances in the cells (see, for example, “Atlas of blood and bone marrow cells” edited by G.I. Kozintsa, Moscow: Triada-X, 1998 ., p. 65-73).

After staining, the biochip is rinsed with water or another liquid and dried. Then carry out the preparation of a long-term drug by any known method used when working with cytological preparations. It is possible, in particular, to enclose the biochip in a Canadian balsam or other composition with similar properties, for example, in the preparation liquid and cytological histological preparations “Shandon-Mount” (manufacturer - “Thermo-electron corporation”, USA, Pitsburg).

The result is read, for this an image of spots and background sections of the biochip is obtained (for example, by photographing with a camera mounted on a microscope or other device) and the density of cell binding (the number of cells bound on the biochip surface area of a given area) in the area of the biochip spots is determined and background plots. The process can be automated.

If the biochip was incubated with the cell suspension without any stirring or the appearance of fluid flows, the analysis of the result makes it possible to determine the content in the suspension of cells expressing detectable surface antigens. The binding density of cells in the area of the biochip spot is proportional to the content in the suspension of cells expressing the corresponding surface antigen. When analyzing the result, the ratio of the values of cell binding density in the area of each biochip spot to the maximum possible value of cell binding density under the given conditions (in the area of the positive control spot) is determined. As such, for example, a spot with antibodies specific for the CD45 antigen can act on a biochip intended for the study of leukocytes. The results can be expressed as a percentage (figure 1, 3). These values are numerically well consistent with the actual percentage in the sample of cells having the appropriate antigens, determined using reference methods. In addition to quantifying the density of cell binding, in some cases, its qualitative or semi-quantitative determination can be used. In these cases, the assessment of the content of cells having detectable antigens in the sample will also be qualitative or semi-quantitative.

Depending on the goals and objectives of the study, the binding density of cells in all spots of the biochip or only in some spots can be determined.

A morphological study of cells bound in the area of each biochip spot is performed (Figs. 1, 2). It is carried out by direct microscopic examination of cells bound in the area of the biochip spots or by studying the images obtained by microphotography of the spots. Microscopic examination can be performed both with the use of lenses that do not come into contact with the surface of the drug, and with the use of immersion (oil) lenses. In this case, multiple microscopic examination of the drug using immersion lenses without any damage to the drug and contamination of the lenses is possible. The image of biochip spots and cells bound in them can also be obtained, for example, using projection equipment, which in some cases is more preferable.

When conducting a morphological study of cells bound to the biochip, the signs necessary for their identification (the shape and size of cells and their nuclei, nuclear-cytoplasmic ratio, chromatin structure, presence or absence of nucleoli, presence of perinuclear clearing, color of the nucleus and cytoplasm, presence of vacuoles are determined , granules and other cytoplasmic structures, as well as the presence, localization and nature of staining of any other structures and objects detected by the staining method used). Based on this, it is determined which cells are bound in the area of a particular biochip spot. When analyzing the result in the area with a given area of a spot, the number of bound cells of certain types or cells having any morphological or other characteristic (set of characteristics) can be determined. The ratio of the binding density of cells of one kind or another to the total density of binding of cells in the region of a given spot or to the maximum possible binding density can be determined. This approach allows us to determine the content in the sample of cells belonging to different subpopulations.

If necessary, a morphometric study of the bound cells can be carried out. A morphological or morphometric study of bound cells can be carried out both without automation (for example, by viewing each biochip spot under a microscope or by studying microphotographs of areas of the corresponding spots), and using computer programs that recognize the image and analyze it.

The claimed invention is aimed at improving the accuracy, reliability and diagnostic value of the analysis results.

Examples of the use of the method.

Example 1

Two biochips (No. 1 and No. 2) were used to study lymphocytes isolated from the peripheral blood of patient C., 51 years old, with a diagnosis of chronic B-cell lymphatic leukemia (B-CLL). Cells were resuspended in isosmotic buffer solution, to which EDTA (to a concentration of 0.5 mg / ml) and heat-inactivated human serum (20% by volume) were added.

Biochips with antibodies (IgG) specific for antigens were used: CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD16, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD36, CD38, CD41, CD44, CD45, CD45RA, CD56, CD71, CD72, CD95, CD98, HLA-DR, IgM. The biochip is a substrate made of a transparent material, in the specified areas of which these antibodies were immobilized.

Using biochip No. 1, the study was performed by the claimed method, and using biochip No. 2, the study was carried out by the method taken as a prototype.

Prior to the study, biochips were fixed in transparent cuvettes and treated with a protein solution that blocks nonspecific binding sites and reduces the strength of nonspecific binding of cells to the substrate. After performing this treatment, the biochips were washed with a detergent solution and a buffer solution.

1000 μl of a cell suspension with a cell concentration of 10 ⁶ cells / ml was added to each cuvette. Incubation of biochips with cell suspension was carried out for 60 minutes without any stirring. Then, the biochips were rinsed several times with isotonic buffer solution to wash off cells that did not bind to immobilized antibodies. The quality of the wash was controlled using microscopy (an inverted microscope was used). Washing was considered performed when no bound cells remained in those parts of the biochip surface where there were no immobilized antibodies. The washing solution was drained from the cell with biochip No. 1 and its remaining drops removed from the walls of the cell with an automatic pipette without touching the surface of the biochip (there were also solution drops on it). This manipulation was performed in a room where 100% humidity was maintained. The time taken to complete this manipulation was 2 minutes. After that, 1 ml of methanol was added to the cuvette with biochip No. 1 and incubated for 15 minutes. After that, methanol was drained.

Washing liquid was poured from the cell with biochip No. 2. After that, the biochip was dried in air for 30 minutes, and then fixed with methanol for 15 minutes and dried again.

Both biochips were stained according to Romanovsky-Giemsa. For this, a diluted solution of azure II and eosin was poured into the cuvettes and incubated for 35 minutes. Then the dye solution was drained, and the cells with biochips were rinsed with water and dried. Biochips were removed from the cuvettes and long-term preparations were prepared from them. For this, each of the biochips was placed on a glass slide, where a drop of liquid was previously applied to prepare long-term histological preparations (Shandon-Mount; manufacturer - Thermo-electron corporation, Pittsburgh, USA). The biochip was pressed lightly, another drop of this liquid was applied on top of it, covered with a coverslip and placed under a press until it hardened.

Then, each spot of the biochip was photographed using a camera mounted on a microscope. On the obtained micrographs, 3-4 regions with a given area (in this case 100x100 μm) were selected and cells were counted in each of them. The obtained values were averaged. The average cell binding densities obtained for each of the biochip spots were expressed as a percentage. The average cell binding density in the area of the biochip spot with antibodies specific for the CD45 antigen was taken as 100% (this antigen is present on the surface of all types of white blood cells). The results obtained (biochip No. 1) are given in figure 1.

Then a morphological study of the bound cells was performed. For this, each biochip spot was viewed at high magnification (a lens with a magnification of x100 was used). The preparation of a long-term preparation from a biochip made it possible to use an immersion lens without damaging the bound cells and their mechanical separation from the biochip surface. Using a microscope-mounted camera, micrographs of cells bound in each spot of the biochip were obtained. Examples are given in figure 2. Cells stained on biochip No. 1 (Fig.2-a) well preserved the shape and size, the shape of the nucleus, the structure of chromatin and other morphological features typical of mature lymphocytes. Cells located on the biochip No. 2 (Fig.2-b) were deformed. A significant part of the cells had an altered shape of the nuclei. Part of the cells was destroyed. Some cells looked fused together.

Thus, the use of the claimed method allows to obtain significantly more adequate results compared to the method taken as a prototype.

Example 2

Using a biochip, lymphocytes isolated from the peripheral blood of a patient B., 77 years old, suffering from hairy cell leukemia (ON) were examined. We used exactly the same biochip as in Example 1. Preparation of the biochip for analysis, incubation with a cell suspension and washing from cells that did not bind to antibodies were performed as described in Example 1.

After completion of washing, the buffer solution was drained from the cuvette. Drops of liquid were removed with a pipette from the walls of the cell without touching the biochip. This procedure was carried out at normal air humidity (about 70%), its implementation time was 20 seconds. After this, staining of cells bound to the biochip was carried out according to Pappenheim. For this, immediately after eliminating the excess washing liquid, a May-Grunwald solution (a solution of eosin and methylene blue in methanol) was poured into the cuvette and incubated for 5 minutes. With this treatment, fixation was combined with the first staining step. After this, the biochip was rinsed with distilled water, a diluted solution of azure II and eosin was poured into the cuvette and incubated for 30 minutes. Then the dye solution was drained, and the biochip was again rinsed with water.

Preparation of a long-term preparation from a biochip was carried out as described in example 1.

As in example 1, the image of biochip spots was obtained, the density of cell binding was determined, and the percentage of cells (of all types) having each of the determined antigens was estimated. The results are shown in figure 3.

A morphological study of bound cells was performed. The cell structure was well preserved (figure 4). There were no signs of deformation, fragmentation of nuclei, or destruction of cells. When stained according to Pappenheim, all the morphological signs of the cells were well defined. A significant part of the cells bound to the biochip was represented by round-shaped lymphocytes, with a round or bean-shaped nucleus with sparse chromatin, pale cytoplasm, which has thin outgrowths resembling villi, which is typical for hairy cell leukemia cells.

Translation of a fragment of an article - description of an analogue.

The reactivity of CD antigens, determined by flow cytometry, was verified by binding of cells to the same antibodies immobilized on a plastic biochip. This biochip was created by staining individual antibodies on the surface of a Falcon polystyrene Petri dish - 1034 (65 x 15 mm) in 10 mm increments. On 1 cup, 16 spots of antibodies could be applied. Each spot was moistened with 1 μl of PBS before applying 0.1 μg of antibody in 0.1% BSA-PBS [bovine serum albumin (BSA) solution in phosphate buffer (PBS)]. Antibodies contacted the plastic surface for 1 hour. Then the biochip was rinsed with 0.1% BSA - PBS. Then incubated for 1 hour in 1% BSA-PBS. Cells were resuspended in small aliquots (less than 5 μl) in 0.1% BSA - PBS. Cell binding [on such a surface] was convenient for light microscopy and photography. This method was also used to determine fusin, integrin β7, CD47, CD62L, CD62E, CD72, CD93, CD99R, CD114, CD121a, CDwl25 and CDwl31 antigens.

Claims (1)

A method for studying cells using a biochip containing immobilized antibodies capable of binding to surface cell antigens, including incubating the biochip with a suspension of cells, washing the biochip from non-specifically bound cells, fixing and staining related cells, obtaining an enlarged image of the surface areas of the biochip, assessing cell binding density in one or more sites, the analysis of the image of the bound cells, characterized in that the fixation is carried out immediately after it is removed I in laundering liquid from the surface of the biochip or removal is performed before fixation in laundering liquid at a humidity of 100% for the time during which there is no occurrence of gross morphological changes in cells.

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