RU2393216C1 - Method for combined immunobiological analysis of cells using biochip - Google Patents

Abstract

FIELD: chemistry; biochemistry. ^ SUBSTANCE: method for combined immunobiological analysis of cells using a biochip involves incubation of the biochip which contains immobilised antibodies, with suspension of cells, washing the biochip from non-bonded cells, determination of coexpression of antigens on the bonded cells. The obtained result is assessed by determining presence of bonded cells in the region of the stain of the biochip and bonding density of cells and interpretation of the obtained result. Coexpression of antigens on cells bonded to the biochip is determined by carrying out one or more immunocytochemical reactions. When reading out the result, morphological analysis of cells bonded to the biochip is also carried out and presence and character of colouring of cells and their components with the reaction product are determined. ^ EFFECT: use of the disclosed method provides high reliability and information content of analysis. ^ 9 cl, 6 dwg, 2 ex

Description

The invention relates to medicine and can be used for diagnostic purposes in hematology, oncology and other branches of medicine, as well as in veterinary medicine and biology.

A known method for determining surface cell antigens using biochips (see Liu AY Differential Expression of Cell Surface Molecules in Prostate Cancer Cells // Cancer Research. 2000, Vol.60., P. 3429-3434), taken as an analogue, which consists in that the biochip (which is a substrate with antibodies immobilized in strictly defined areas, each of which is specific to a specific cellular surface antigen) is incubated with a suspension of cells. Cells with corresponding antigens on their surface bind to antibodies immobilized in strictly defined areas of the biochip (spots), the remaining cells do not specifically bind to its surface. Then the biochip is washed from non-specifically bound cells by rinsing with a buffer solution. As a result, only those cells that bind to antibodies remain on the biochip surface. The result is read out, determining in which spots of the biochip cell binding occurred. The method allows to simultaneously determine on different cells a large number of different surface antigens. The maximum possible panel of antibodies, which can be placed on one biochip, is limited only by the size of the substrate and the size of the areas (spots) of the biochip in which antibodies are immobilized.

The disadvantage of this method is that on each individual cell upon the fact of binding in one or another spot of the biochip, only one antigen can be determined. The inability to determine a greater number of antigens (co-expression) on each individual cell reduces the diagnostic value of the analysis and can cause errors. In addition, the method does not provide for morphological studies of cells, which does not allow them to be identified.

A known method for the study of cells using immunological biochips (see Belov L, de la Vega O, dos Remedies CG, Mulligan SP, Christopherson RI Immunophenotyping of leukemias using a cluster of differentiation antibody microarray // Cancer research, - 2001. - Vol.61 - P.4483-4489), which consists in the fact that a biochip containing immobilized antibodies is 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. After this, the biochip is incubated with a solution of antibodies conjugated with fluorochrome, and washing is carried out. The density of the filling of the biochip spots with the bound cells is determined. To determine coexpression of antigens, a biochip is examined, for example, using a luminescent microscope. Moreover, the presence (or absence) of fluorescent cells in the biochip spots is determined and their number is estimated. Then carry out the interpretation of the result.

The disadvantage of this method, taken as a prototype, is the need to use expensive equipment.

The disadvantage is the very need for ultraviolet radiation, due to the use of fluorescent labels. Under the influence of ultraviolet radiation, there is a rapid decrease in the fluorescence of labels (“fading”) due to their destruction. The biochip cannot be re-examined after any long time.

In addition, when reading the result, it is impossible to conduct a morphological study of cells (it is only possible to establish the localization of the label and, in some cases, assess cell sizes). This does not allow to identify cells on the basis of morphological features and dramatically reduces the information content of the method.

All of the above limits the use of this method.

The objective of the claimed invention is: to increase the reliability and information content of the analysis while reducing its cost and the ability to perform without the use of expensive equipment.

The problem is solved due to the fact that according to the method of combined immunological research of cells using a biochip, including incubating a biochip containing immobilized antibodies with a suspension of cells, washing the biochip from unbound cells, determining coexpression of antigens on connected cells, reading the result, including determining the presence binding of cells in the area of biochip spots and determining the density of cell binding, and interpretation of the result, determination coexpression of antigens on biochip-related cells is performed by performing one or more immunocytochemical reactions, while reading the result, a morphological study of cells associated with the biochip is additionally carried out, and the presence and nature of staining of the cells and their components with the reaction product is determined.

An additional staining of the cells is carried out, which does not interfere with the detection of the product (s) of the enzymatic reaction.

To study one sample of a cell suspension, several biochips are used, on which immunocytochemical analysis of cells is carried out using antibodies specific for different antigens.

A morphometric study of cells associated with the biochip is carried out.

During the analysis, one or more additional control biochips are used.

The biochip is treated with one or more solutions containing one or more substances that cause an increase in the strength of the binding of cells to the surface of the biochip.

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

A biochip or a long-term preparation made from it is marked, making it easier to find the right spots.

When conducting the analysis, one or several additional biochips are used, on which stained cells are stained for their subsequent morphological examination, while the immunocytochemical reaction is not carried out.

Using the claimed method allows to determine the presence or absence of coexpression of antigens on each individual cell associated with the biochip. In this case, one antigen is determined more than during immunocytochemical studies without the use of a biochip. An immunocytochemical (enzyme immunoassay) study of cells associated with a biochip can be performed using the direct method (see, for example, Petrov S.V., Raykhlin I.T. Guidelines for the immunocytochemical diagnosis of human tumors, 3rd edition, revised, Kazan: Title, 2004 .-- p.21). Such a study is the least time-consuming and requires less time and reagents, but gives less intense staining of cells with reaction products. An immunocytochemical study of biochip-related cells can also be performed using any indirect immunochemical method or using the EPOS, En Vision, and CSA methods (see, for example, Petrov S.V., Raykhlin I.T. Guidelines for the immunocytochemical diagnosis of human tumors, edition 3rd, revised, Kazan: Title, 2004. - p.22-25). In this case, the implementation of this stage of the analysis is longer and more laborious, but due to the greater intensity of staining of the cells with the reaction product, sensitivity increases, which allows the determination of weakly expressed antigens. In some cases, it is necessary to determine the presence or absence of coexpression not of two, but, for example, three antigens on each cell. In this case, double cytochemical staining is used, which is based on a different color visualization of two different antigens (the third antigen is determined by the fact that the cell binds in the region of one or another biochip spot). For this, two immunocytochemical reactions are carried out (possible combinations of immunocytochemical methods: direct / direct, direct / indirect, indirect / indirect method, double EPOS method, double En-Vision method). (See, for example, Petrov S.V., Raykhlin I.T. Guidelines for the immunocytochemical diagnosis of human tumors, 3rd edition, revised, Kazan: Title, 2004. - p.25-27). By the same principle, it is possible to determine a larger number of antigens.

The use of enzyme-linked immunosorbent reactions eliminates the disadvantages characteristic of immunofluorescence analysis: fading labels, the impossibility of re-conducting the study and others. Moreover, to carry out the research by the proposed method, expensive equipment is not required. The study of cells on the biochip can be carried out using a conventional light microscope. Microscopic examination can be carried out repeatedly and after a long time after the completion of the immunocytochemical reaction without distorting the results.

Morphological examination can determine the shape and size of cells and their nuclei and other structures, as well as other signs. Not only the presence of staining of the cells with the reaction product is determined, but also its nature (staining intensity, localization of the stained product), which makes it possible to evaluate on which particular cell structures the determined antigen is located and estimate its quantity.

For a more complete morphological study, additional staining of cells associated with the biochip can be carried out, not interfering with the detection of the product (s) of the immunocytochemical reaction. For example, when using 3-diaminobenzidine tetrachloride (DAB) as a chromogen, Romanovsky-Giemsa staining or hematoxylin staining is possible (FIG. 2). As a result, the information content of the study is significantly increased. It becomes possible to identify various subpopulations among cells bound in any of the biochip spots (and, therefore, having one or another surface antigen). In this case, cell subpopulations can be determined both on the basis of the results of determining coexpression of antigens using an immunocytochemical reaction, and on the basis of the results of a morphological study. When processing the results, the content of cells belonging to a particular subpopulation can be quantified. Their proportion from the number of cells bound in a given biochip stain or from the total number of cells can be determined.

In some cases, it is necessary to determine several variants of coexpression of antigens, which cannot be performed on one biochip. In this case, several biochips can be used on which immunocytochemical reactions are carried out (one or several) using several enzyme-conjugated antibodies specific for different antigens.

A morphometric study of bound cells can be carried out, which significantly increases the information content of the analysis. This approach can be used for research purposes.

To eliminate errors during the analysis of the same sample, additional control biochips (one or several) can be used. For example, biochips containing antibodies specific for other epitopes of antigens detected by the first biochip can be used. In this case, a comparison of the results obtained using the main and control biochips (one or more) allows us to exclude errors associated with the cross-reactivity of antibodies, as well as errors associated with a loss of activity of the antibodies used. To eliminate errors associated with immunocytochemical staining of the drug, control biochips can be used on which the bound cells are fixed in a different way (for example, fixation with absolute ethanol instead of acetone or vice versa) or a different cytochemical reaction or a different chromogen. It is also possible to carry out various positive and negative controls to determine the specificity of binding to cellular antigens of antibodies used in the cytochemical reaction (s).

After washing from cells that did not bind to antibodies, in some cases, the biochip can be treated with substances that increase the binding strength of the remaining cells to the biochip surface. This increases the accuracy of the analysis, since it allows to prevent separation of cells during subsequent manipulations and to eliminate false negative results. Using this approach also increases the sensitivity of the analysis, since it allows you to examine cells weakly bound to immobilized antibodies (for example, due to the low expression of the corresponding antigens).

The preparation of a long-term preparation from a biochip allows a morphological study of coupled cells to be carried out repeatedly using immersion lenses without contaminating the latter and without damaging the surface of the biochip and cells connected to it. This approach is very useful, for example, in cases where for the correct interpretation of the result it may be necessary to view the drug by several specialists. In addition, this allows a comparative study of cells of the same patient, subjected to analysis using different biochips at different times. This can be very useful, for example, for assessing the dynamics of a disease or for evaluating treatment outcomes. Preparation of a long-term preparation allows storing the biochip for an unlimited time and makes it much more resistant to various damages. It is possible to prepare a long-term drug in various ways. It should be borne in mind that the use of this approach is possible if the substances used for this do not lead to the dissolution of the product (s) of the enzymatic reaction.

A marking can be applied to the biochip or a long-term preparation made from it (Fig. 6), which makes it easier to find and identify spots and avoids possible errors, for example, when the result is read out automatically.

In some cases, additional staining of the biochip with the most preferred method for morphological study of cells prevents the detection of the reaction product. For example, when using not DAB, but some other chromogens, Romanovsky-Giemsa staining prevents the detection of the reaction product. In these cases, during the analysis, one or several additional biochips can be used, on which only staining of the bound cells is necessary, which is necessary for carrying out a morphological study. Immunocytochemical reactions are not carried out. The results of the study of cells obtained using the additional biochip (biochips) are compared with the results of the study of cells obtained using the biochip (biochips), on which the cytochemical reaction was carried out. This increases the reliability of the analysis and avoids possible contradictions between the results of immunophenotyping and the results of morphological studies.

The proposed method can be used in various modifications, suitable for clinical research, as well as for solving various research and other tasks.

The claimed method is illustrated by drawings (Fig.1-6).

Figure 1 is a micrograph of a fragment of a biochip, including spots with immobilized antibodies. In the area of spots, cells isolated from the blood of a patient with chronic B-lymphocytic leukemia (B-CLL) were bound. The density of cell binding in the stain region with different antibodies is not the same. An increase of 17.5 times.

Fig. 2 micrographs of cells of a patient suffering from chronic B-lymphocytic leukemia (B-CLL) bound on a biochip in the spot region with anti-CD23 antibodies. The expression of the CD19 antigen was immunocytochemically determined. The nature of cell staining with the reaction product indicates the surface localization of the CD19 antigen. Thus, co-expression of CD19 / CD23, characteristic of cells of chronic B-lymphocytic leukemia, is detected. Cell nuclei were stained with hematoxylin. 400x magnification.

Figure 3 is a diagram showing the result of determining the content in the test suspension of cells expressing various surface antigens. Using a biochip, we studied cells isolated from the peripheral blood of a patient with chronic B-lymphocytic leukemia (B-CLL).

Figure 4 is a diagram showing the result of determining the content in the test suspension of cells expressing various surface antigens. Using a biochip, we studied cells isolated from the peripheral blood of a healthy person.

5 is a micrograph of cells of a patient with chronic B-lymphocytic leukemia (B-CLL), connected on an additional biochip in the spot region with anti-CD19 antibodies. Coloring according to Romanovsky-Giemsa. 900x magnification.

Fig.6 is a photograph of a section of a long-term preparation made from a biochip, on which a marking is applied before the manual reading of the result, making it easier to find the right spots. The grid layout divides the biochip spots into groups in the form of “rows” and “columns”. This avoids errors when sequentially photographing biochip spots through a microscope. In this case, the marking is applied manually. 3 times increase.

The claimed method is as follows. Before the analysis, the biochip (which is a transparent plate on which antibodies specific for cell surface antigens are immobilized in strictly defined areas (spots)) are fixed in a container, for example, in a Petri dish, container or cuvette. Instead, the biochip can be fixed in the flow chamber (see Shishkin A.V., Shmyrev I.I., Kuznetsova S.A., Ovchinina N.G., Butylin A.A., Ataullakhanov F.I., Vorobyov A . I. Immunological biochips for the study of human red blood cells, Biological membranes, 2008, Volume 25, No. 4, pp. 267-276). Biochip is incubated with the studied cell suspension. 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 to eliminate cells that are not bound to antibodies. If the biochip is in the flow chamber, washing is carried out by passing a wash solution stream over the surface of the biochip at a given speed. If the biochip is in a Petri dish, cuvette (container) or other container, it can be washed by rinsing with a washing solution or by repeatedly placing (immersing) in this solution. After washing in the area of the biochip spots, only those cells that have the corresponding surface antigens remain bound. Quality control of washing is carried out using microscopic examination. Washing is considered complete if there are no bound cells outside the biochip spots (in the background areas).

After completion of washing from cells that did not adhere to antibodies, in some cases (for example, when using flow chambers with a narrow capillary to prevent separation of cells when the capillary is filled with air before removing the biochip), the biochip can be treated with solutions of substances that increase the binding strength of the remaining cells to it surface. When working with leukocytes using a biochip made on a plastic substrate, for example, incubation (10-20 minutes) with a solution containing calcium and magnesium salts, whose ions participate in the processes of cell adhesion to solid surfaces, can be used. When choosing the substances used, it should be borne in mind that carrying out such processing should not impede the implementation of the subsequent stages of the study, primarily the immunocytochemical reaction (s). After this treatment, the biochip is washed with isotonic buffer solution.

Then the biochip is removed from the tank (or flow chamber), where the previous steps of the analysis were performed. If reuse of the container is not provided, the biochip can be removed from it and all subsequent manipulations related to the processing of the biochip with the solutions used can be carried out in it.

Then the cells bound to the biochip are fixed. For this, for example, the biochip is dried (the optimal drying time in air is 1 hour) and is treated in absolute acetone at + 4 ° C for 1-3 minutes. It is also possible to fix the biochip with bound cells with absolute ethanol (10-15 minutes), followed by drying. When using the chosen fixation method, the biochip substrate should not dissolve or lose its transparency under the action of the fixing substance.

Next, an immunocytochemical reaction (s) is carried out by any known method. In this case, it is possible both to carry out one immunocytochemical reaction and to carry out double immunocytochemical staining (see, for example, Petrov S.V., Raykhlin I.T. Manual for immunocytochemical diagnosis of human tumors, 3rd edition, revised, Kazan: Title, 2004 .-- p.21-27). It is possible to perform a larger number of immunocytochemical reactions on cells associated with a single biochip, but this approach is unnecessarily time-consuming.

In the case of using the immunocytochemical method of EnVision to carry out one immunocytochemical reaction, the sequence of actions will be as follows.

A 3% hydrogen peroxide solution is treated for 10 minutes, after which the biochip is washed twice with buffer solution (TBS) for 5 minutes. Then, a biochip with a solution of non-immune serum is incubated for 20 minutes to block non-specific binding of antibodies. Then excess serum is removed and incubated with a solution of primary antibodies for 40 minutes, after completion of the incubation, the biochip is rinsed with buffer and washed twice with TBS (5 minutes each). Then, the remaining liquid is removed and EnVision reagent, which is a polymer macromolecule conjugated with secondary antibody molecules and enzyme molecules, in this case horseradish peroxidase, is added. An incubation is carried out for 40 minutes. Then the biochip is rinsed with buffer and washed twice with TBS (5 minutes each). After that, the biochip is incubated with a solution of DAB, which is carried out in the dark for 5-10 minutes (when determining some antigens, the optimal incubation time may differ up or down). The biochip is washed with distilled water and dried if necessary. It is possible to carry out additional staining of cells, for example, with hematoxylin or according to Romanovsky-Giemsa (in the latter case, fixation with acetone is preferable). Such staining of the cells in this case does not prevent the detection of the product of the immunocytochemical reaction (when using the DAB chromogen), if the “repainting of the drug” has not occurred. It should be noted that during additional Romanovsky-Giemsa staining, it is necessary to carefully maintain the established time for processing the biochip with dye, since “repainting” begins to interfere with the detection of the reaction product. The optimal incubation time of the biochip with dye (s) is selected experimentally, since it can be different for different batches of dye, and also depends on the concentration of the dye solution. After additional staining, the biochip is rinsed with distilled water.

In some cases, it may be necessary to determine the coexpression of several pairs (or groups) of antigens, which cannot be performed on a single biochip. In this case, several biochips can be used on which different antigens are immunocytochemically determined. In this case, any immunocytochemical method can also be used. If necessary, additional staining can also be performed.

Then, a long-term preparation can be prepared from the biochip. To do this, a drop of Canadian balsam (or other substance with similar properties, for example, a liquid for the preparation of histological preparations "Shandon-Mount"; manufacturer - "Thermo-electron corporation", USA, is applied to the surface of a glass slide (or a transparent plate of a different material); Pitsburg). A biochip is placed on top (with the side of the bound cells up) and pressed so that there are no air bubbles between it and the glass. Then another portion of Canadian balsam is applied to the surface of the biochip. On top impose and in the same way press the coverslip. The drug is placed under a load or press until the Canadian balsam hardens. The preparation of a long-term preparation is possible if the substances used for this do not lead to the dissolution of the product (s) of the enzymatic reaction. For example, the above-described variant of the methodology for preparing a long-term drug can be applied using DAB as a chromogen. It is possible to prepare a long-term drug in other ways.

Further, if necessary, a marking can be applied to the biochip or a long-term preparation made from it (Fig. 6), which facilitates the finding of the necessary spots during the manual reading of the result. For example, in the simplest case, lines can be drawn dividing the biochip spots into groups (for example, rows and columns), which subsequently avoids errors when sequentially photographing biochip spots through a microscope.

Then, the result is read, for this an image of spots and background sections of the biochip is obtained (Fig. 1), (for example, in the simplest case by photographing through a microscope), and the cell binding density (the number of cells bound on the biochip surface area of a given area) is determined in the region biochip spots and background areas. Depending on the objectives of the analysis, a qualitative, semi-quantitative or quantitative determination of the content in the test suspension of cells having these surface antigens can be carried out. For the quantitative determination of the cell binding density in the simplest case, direct cell counting in the region of specified sections of the biochip surface can be used. For example, in photographs of biochip spots, sections of a given area where cells are counted can be selected. This process can be automated.

Next, the content in the test suspension of cells having detectable (using a biochip) surface antigens is determined (FIGS. 3, 4). To this end, the binding density of cells in the area of a spot is expressed as a percentage of the maximum binding density that is taken as 100% under the given conditions (determined, for example, by calculation) or relative to the density of binding of cells in the area of a spot with antibodies that can bind to all studied cells (for example, with anti-CD45 antibodies in the study of leukocyte suspensions). The values obtained in this case are in good agreement with the actual content in the suspension of cells having detectable surface antigens. The use of this approach is possible if the incubation of the biochip with a cell suspension was carried out without stirring or supplying a fluid flow, and the sizes of cells belonging to different subpopulations are quite close.

Next, a morphological study of cells bound in the area of each biochip spot is carried out. In this case, the morphological characters necessary for the identification of cells are determined, their staining is determined by the product of the immunocytochemical reaction (s), and its features (intensity, localization) are evaluated. A morphological study can be performed both without automation (for example, by viewing the preparation under a microscope or by studying micrographs of cells bound in the area of the biochip spots) and using automation (for example, using computer programs that recognize the image and analyze it). If necessary, a morphometric study of the bound cells can also be carried out. If a long-term preparation was prepared from a biochip, microscopic examination is possible both with the use of lenses that do not come into contact with the surface of the preparation, and with the use of immersion lenses.

During microscopic examination, in each spot of the biochip in areas of a given area, the number of cells stained with the reaction product and, therefore, expressing the corresponding antigen together with the antigen determined by cell binding in this spot of the biochip can be determined. Similarly, the number of cells having any other distinctive (morphological, immunocytochemical or morphometric) trait can be determined. The fraction of such cells from the number of cells bound in a given spot in a given area can be determined, or their percentage in the test sample of the cell suspension can be determined by calculation.

In some cases, the study requires the use of additional biochips.

1) In some cases, it is necessary to use additional control biochips (one or several), on which only the cells are stained, necessary to perform their morphological studies. An immunocytochemical reaction is not carried out. This approach can be used, for example, if the staining using the most suitable method for morphological investigation prevents the detection of the reaction product formed when using the selected chromogen, or when several types of additional coloring are required. When processing the results, data obtained using the main and additional biochips is compared. Conducting a morphological study on an additional biochip (biochips) increases the reliability of the analysis and avoids possible contradictions.

2) Additional biochips can be used for control. Moreover, the number of used control biochips and the conditions for conducting control studies depend on the tasks set (assessment of the specificity of cell binding to antibodies immobilized on the biochip, study reproducibility, control of immunocytochemical reactions, positive and negative control during immunocytochemical reactions, and others) and can be individual in each case.

The claimed invention is aimed at improving the reliability of the method, reducing the cost of analysis, achieving the ability to perform research in laboratories that do not have expensive equipment, increasing the information content and diagnostic value of the analysis results, as well as achieving the possibility of long-term storage of biochips on which the analysis was performed for repeated microscopic studies.

Examples of the use of the method.

Example 1

The cells of patient A., 61 years old, with mature cell lymphoproliferative disease accompanied by bone marrow damage, were examined. When examining a blood smear, an increased content of mature lymphocytes was noted. It was necessary to determine the T- or B-cell nature of the tumor and determine its nosological form. To do this, it was necessary to determine the immunophenotype of tumor cells.

We used a set of 3 biochips containing immobilized antibodies (IgG) specific for antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD16, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31 , CD36, CD38, CD41, CD44, CD45, CD45RA, CD56, CD71, CD72, CD95, CD98, HLA-DR, IgM.

The leukocyte fraction of the cells was isolated by centrifugation on a density gradient. The isolated cells were resuspended in the working solution. Biochips were fixed in cuvettes. The cuvettes were filled with cell suspension and incubated for 40 minutes without any stirring.

Then the biochips (which remained in the cuvettes) were rinsed with a washing solution to eliminate cells that did not bind to antibodies. Quality control washing was carried out using microscopy. Biochips were treated with absolute ethanol for 10 minutes and dried.

Next, an immunocytochemical reaction was carried out using the EnVision method (the course of the reaction is described above) according to the following procedure: 1) treatment with a 3% hydrogen peroxide solution for 10 minutes; 2) rinsing with buffer (TBS) followed by washing twice with TBS for 5 minutes; 3) incubation with a solution of primary antibodies specific for the antigen to be detected for 40 minutes; 4) rinsing with buffer (TBS) followed by washing twice with TBS for 5 minutes; 5) removal of the remainder of the liquid; 6) incubation of the biochip with EnVision reagent ("Daco", USA) for 40 minutes; 7) incubation of the biochip with a solution of the DAB chromogen in the dark for 10 minutes; 8) washing the biochip with distilled water and drying.

On cells associated with biochip No. 1, immunocytochemical determination of CD19 antigens was performed.

Immunocytochemical determination of CD2 antigens was performed on cells associated with biochip No. 2.

For staining of nuclei in bound cells, biochips No. 1 and No. 2 were additionally treated with hematoxylin.

Biochip No. 3 was not treated with any reagents necessary for immunocytochemical studies. Instead, after washing from cells that did not bind to antibodies, it was fixed with methanol (15 minutes) and stained according to Romanovsky-Giemsa, for which it was treated with a solution containing azure and eosin for 40 minutes.

After that, long-term preparations were prepared from biochips and microscopic examination was performed.

The density of cell binding was determined in the area of various spots of the biochip, based on which the percentage of cells was determined. The cell binding density was determined by direct counting. For this, the number of cells bound in areas of a given area was counted in each spot of the biochip. Counting was performed in at least 3 sections of 100 × 100 μm, the obtained values were averaged. The cell binding density in each spot was expressed as a percentage. The cell binding density in the spot region with anti-CD45 antibodies (11500 cells / mm ² ) was taken as 100%. The results are shown in figure 3.

In addition to the control spots with anti-CD44 and anti-CD45 antibodies (where all leukocytes can bind), the highest cell binding density was observed in the area of biochip spots with antibodies specific for antigens: CD5, CD19, CD20, CD21, CD22, CD23, CD27, CD29 , CD29, CD31, CD72, CD98, HLA-DR. In a morphological study of cells bound in the area of these spots, it was found that almost all of them are mature lymphocytes.

On biochip No. 1, the accumulation by the cells (all or most of them) of the stained product of the enzymatic reaction was observed in the stain region with antibodies specific for antigens: CD5, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD44, CD45, CD45RA, CD72, CD98, HLA-DR. This indicates the joint expression on the same cells (co-expression) of the CD19 antigen with the antigens CD5, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD44, CD45, CD45RA, CD72, CD98, HLA-DR. Figure 2 is a photomicrograph illustrating an example of determining the co-expression of antigens CD19 and CD23 (co-expression of CD19 / CD23).

On biochip No. 2, the accumulation by the cells of the stained product of the enzymatic reaction was observed in the stain region with antibodies specific for the antigens CD2, CD3, CD4, CD8 (on all cells connected in these spots) and CD7 (on part of the cells), in addition, a small part was stained cells bound to stain with antibodies anti-CD5, anti-CD44, anti-CD45, anti-CD45RA. Thus, co-expression of CD2 / CD3, CD2 / CD4, CD2 / CD5, CD2 / CD7, CD2 / CD8, etc., typical of mature T cells, was noted. Thus, only cells located in the stain region with antibodies specific for the reaction were stained to T-cell antigens and those antigens that are found on different types of cells. The density of cell binding in the area with antibodies specific for T-cell antigens was low, indicating a low content of T-lymphocytes in the test sample (figure 3). These cells are normal T lymphocytes, which are present in small numbers against the background of a much larger number of tumor cells. The cell binding density in the spot region with antibodies specific for the CD5 antigen was high, but only a small fraction of these cells accumulated a stained product. Consequently, most of the cells bound in this spot of the biochip were not T-lymphocytes. This indicates their aberrant immunophenotype. The reaction product also stained some cells bound in the spot region with anti-CD31 antibodies (CD31 antigen is present on part of T-lymphocytes), and single cells in the spot region with anti-CD16 and anti-CD56 antibodies (CD2 antigen is present on part of NK- cells).

Thus, this tumor is B-cell. The presence of coexpression of CD5 / CD19 / CD23 in the absence of lymphocyte expression of the CD10 antigen is typical for cells of chronic B-lymphocytic leukemia. The results were subsequently confirmed clinically.

Example 2

The cells of a healthy B. donor, 29 years old, were examined. The aim of the study was to verify the binding specificity of various subpopulations of lymphocytes (T, B, and NK cells). A set of 3 biochips was used, the same as in example 1. The selection of leukocytes from peripheral blood, their incubation with a biochip, washing of the biochip and fixation were carried out in the same manner as described in example 1.

After that, an immunocytochemical reaction was carried out on each of the biochips.

On cells linked to biochip No. 1, the expression of the CD19 antigen was immunocytochemically determined.

On cells linked to biochip No. 2, the expression of CD16 antigen was immunocytochemically determined.

On cells bound on biochip No. 3, expression of the CD2 antigen was immunocytochemically determined.

The methodology for carrying out immunocytochemical reactions by the EnVision method was the same as described in example 1.

In the same manner as in Example 1, the binding density of cells in the region of various biochip spots was determined, based on which the percentage of cells expressing various antigens in the test sample was determined. The results are shown in figure 4. They are typical for indicators of a healthy person.

Microscopic examination determined the accumulation of the product of the enzymatic reaction on cells located in different spots of the studied biochips.

On biochip No. 1, the accumulation by the cells of the stained product of the enzymatic reaction was observed in the stain region with antibodies specific for the CD19, CD20, CD21, CD22, CD38, CD45RA, CD72, CD98, HLA-DR antigens, which is typical for B-lymphocytes, as well as in stain areas with antibodies specific for the CD44, CD45 antigens present on all white blood cells. In the area of spots with antibodies specific for CD19, CD20, CD21, CD22, CD72 antigens, almost all cells were stained with the reaction product, and in the other biochip spots listed above, only part of the bound cells was stained.

On biochip No. 2, the accumulation by the cells of a stained product of the enzymatic reaction was observed in the stain region with antibodies specific for CD16, CD56 antigens, which is typical for NK lymphocytes. In this case, all bound cells were stained. In the area of spots with antibodies specific for CD44, CD45, CD7 antigens, part of the cells was stained with the reaction product. This is because these antigens express not only NK lymphocytes, but also other types of cells. A small part of the cells that bind in the stain region with antibodies specific for CD2 and CD7 antigens was also stained. This is also the norm. Thus, co-expression of CD16 / CD56, CD16 / CD44, CD16 / CD45, CD16 / CD2, CD16 / CD7 was detected, which is the norm.

On biochip No. 3, the accumulation of the stained product of the enzymatic reaction by almost all cells was observed in the stain area with antibodies specific for CD2, CD3, CD4, CD5, CD8 antigens. In the area of spots with antibodies specific for CD7, CD27, CD29, CD31, CD44, CD45, CD45RA, CD95 antigens, part of the cells was stained. This is because these antigens express not only T-lymphocytes, but also other types of cells. Thus, co-expression of CD2 / CD3, CD2 / CD4, CD2 / CD5, CD2 / CD7, CD2 / CD8, CD2 / CD27, CD2 / CD29, CD2 / CD31, CD2 / CD44, CD2 / CD45 and others was found that is the norm. The presence of coexpression of CD2 / CD95 in some cells may indicate their activation. Some of the cells that bind in the spot region with anti-CD16 antibodies were also stained. This is due to the expression of the CD2 antigen by NK cells. Thus, as a result of the experiment, data indicating the cross-reactivity of antibodies were not obtained, which indicates the suitability of this batch of biochips for use.

Claims (9)

1. A method of combined immunological research of cells using a biochip, including incubating a biochip containing immobilized antibodies with a suspension of cells, washing the biochip from unbound cells, determining coexpression of antigens on connected cells, reading the result, including determining the presence of cell binding in the area of the biochip spots and determination of cell binding density, and interpretation of the result, characterized in that the determination of coexpression of antigens on the bound x biochip with cells operate by performing one or more of the immunocytochemical reactions when read result is further conducted research related morphological biochip cells and determine the presence and nature of the cells and staining them a reaction product of components. 2. The method according to claim 1, characterized in that they carry out additional staining of the cells, not interfering with the detection of the product (s) of the enzymatic reaction. 3. The method according to claim 1, characterized in that for the study of one sample of a cell suspension using several biochips, which carry out immunocytochemical analysis of cells using antibodies specific to different antigens. 4. The method according to claim 1, characterized in that carry out a morphometric study of cells associated with the biochip. 5. The method according to claim 1, characterized in that during the analysis using one or more additional control biochips. 6. The method according to claim 1, characterized in that the biochip is treated with one or more solutions containing one or more substances that increase the binding strength of cells to the surface of the biochip. 7. The method according to claim 1, characterized in that a long-term preparation for microscopic studies is prepared from a biochip. 8. The method according to claim 1, characterized in that a marking is made on the biochip or a long-term preparation made from it, which facilitates finding the right spots. 9. The method according to claim 1, characterized in that during the analysis, one or more additional biochips are used, on which stained cells are stained for their subsequent morphological studies, while the immunocytochemical reaction is not carried out.

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