RU2302635C1 - Method for measuring mitochondrial lymphocyte activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves measuring succinate dehydrogenase activity with flow laser cytometric approach for detecting lymphocyte populations showing metabolism disorders.

EFFECT: wide diagnostic application field in immunologic and allergologic research.

Description

The invention relates to medicine, and specifically to a method for immunological diagnostics.

The invention can most effectively be used both in fundamental and applied fields of medicine and veterinary medicine, namely immunology and cytochemistry.

A known method of immunological diagnosis: immunophenotyping of lymphocytes using flow laser cytometry [Use of flow cytometry to assess the functional activity of the human immune system. // Manual for laboratory assistants, ed. R.M.Khaitova. M .: SSC RF Institute of Immunology, Ministry of Health of the Russian Federation, 2001, 53 pp.]. The method has a high diagnostic value, allows you to determine the number of cells in various populations of lymphocytes. The disadvantage of this method is that it does not give an idea of the functional state of cells in these populations. In clinical practice, there are situations in which the results of immunological diagnostics that do not meet normative indicators do not yet indicate the presence of pathology and, conversely, normal values of lymphocyte subpopulations can be observed in patients with deviations in the functioning of the immune system.

A known method for assessing the functional state of lymphocytes by conducting a reaction of blast transformation of lymphocytes (RBTL) [Lebedev K.A., Ponyakina I.D. Immune deficiency (detection and treatment). M .: Medical book, 2003, 443 p.]. The essence of the method consists in the detection of mitotic activation of lymphocytes under the influence of stimuli (mitogens and antigens). Depending on the quality of the mitogen used, both the mitotic activity of the entire lymphocyte pool and individual lymphocyte populations can be investigated. The disadvantage of this method is the difference in the results obtained when using various modifications, which complicates their clear interpretation in the clinic. Thus, the lack of standardized technology and the need for sterile work limit the widespread use of this diagnostic method.

A known method for assessing the functional state of lymphocytes by determining the antibody-dependent cell-mediated cytotoxicity of lymphocytes [Lebedev K.A., Ponyakina I.D. Immune deficiency (detection and treatment). M .: Medical book, 2003, 443 p.]. The principle of the method is that the suspension of purified lymphocytes is incubated with the target cells, after which the cytotoxic effect of the lymphocytes is determined by the number of remaining target cells by the release by the lysed target cells of the ⁵¹ Cr radioactive isotope into the culture medium. The disadvantage of this method is that it requires complex equipment and working conditions with isotopes, therefore, they are mainly used in specialized laboratories.

The listed methods for determining the functional state of lymphocytes do not give an idea of their metabolic features, which excludes the possibility of targeted metabolic correction of immune disorders. It is known that the functional activity of cells is directly determined by their energy metabolism.

A known method for evaluating the energy metabolism of peripheral blood lymphocytes, based on determining the activity of the main mitochondrial enzyme - succinate dehydronenase (R. Narcissov. Use of violet para-nitrotetherazolium for quantitative cytochemical determination of human lymphocyte dehydrogenases. // Archive of Anatomy, Histology, Embryology. - 1969. - No. 5. - S.85-91).

This method is selected as a prototype of the claimed invention. The basis of this method is the principle of counting granules - the product of the enzymatic reaction. As an indicator of the enzymatic process, p-nitro-terazolium violet is adopted, which forms large spherical granules of formazan upon restoration in lymphocytes.

Reactions were carried out on blood smears prepared on fat-free glass slides. The smears were dried in air at room temperature for 10-15 minutes. The formulation of the reaction includes three stages: the fixation of smears, a reaction to detect the activity of succinate dehydrogenase, and nuclear dyeing.

The preparations were fixed in a 60% acetone solution saturated with disodium salt of ethylenediaminetetraacetic acid (EDTA) at pH 5.3 at room temperature for 30 seconds. The supernatant is used for fixation with aceontrilone. The latch is stored and used at room temperature. After fixation, the preparations were washed with distilled water and dried at room temperature in air.

The medium for detecting succinate dehydrogenase activity consisted of an incubation medium and a substrate, sodium succinic acid, specific for the enzyme detected. The medium is prepared on phosphate buffer and stored in a refrigerator in a glass bowl for no more than 2 weeks. Preparation of the incubation medium: for 40 ml of phosphate buffer 13 mg of p-nitrotetrazolium violet, 13 mg of trilon B and 540 mg of sodium succinic acid. Preparation of phosphate buffer: 100 ml of phosphate buffer are made up of 20 ml of KH ₂ PO ₄ (9.1 grams per 1 liter of water) and 80 ml of Na ₂ HPO ₄ (11.9 grams per 1 liter of water), pH 7.2-7.4. The reaction is carried out at pH 7.3 and a temperature of 37 ° C for 60 minutes in an aqueous thermostat. After incubation, the smears were washed with water and immersed in a saturated solution of methyl green (nuclear dye) for 15-20 seconds, after which the smears were again washed and dried at room temperature in air.

The finished smears were microscopically under water immersion (400 times magnification) using a Biolam R-16 microscope. The activity of the enzyme in the cell was judged by the number of dark-violet granules of formazan formed during the enzymatic recovery of p-nitrotetrazolium violet. To determine the activity of the enzyme in the lymphocyte population, the number of granules in 50 cells was counted, and then the average number of granules in the lymphocyte was calculated (drawing).

The disadvantage of this method is the inability to assess the functional state of the mitochondrial apparatus in individual populations of lymphocytes. Such disadvantages limit the diagnostic capabilities of this method in immunological and allergological diagnostics, since they do not allow to identify lymphocyte populations with impaired metabolism.

The objective of this study was to create a method for measuring mitochondrial activity in individual populations of lymphocytes.

The problem is solved by measuring the activity of succinate dehydrogenase in lymphocytes by incubating permeabilized cells in an incubation medium consisting of a substrate specific for this enzyme - succinic acid, phosphate buffer, p-nitrotetrazolium violet and Trilon B and analyzing the result of the cytochemical reaction using instrumental of the method, venous blood lymphoconcentrate treated with monoclonal antibodies with a fluorescent label is used for analysis, moreover, as e instrumentally using laser flow cytometry, wherein the indicator of mitochondrial activity in isolated populations is determined using the formula:

MA = KBS1 / KBS2 · 100%, where

MA - mitochondrial activity,

KBS1 - the average coefficient of intensity of lateral light scattering in a population of cells before the cytochemical reaction,

KBS2 is the average coefficient of lateral light scattering intensity in a cell population after a cytochemical reaction, while for a CD3 + (T cell) population, a decrease in MA below 152.7% indicates mitochondrial T cell deficiency, an increase in MA above 175.9% indicates mitochondrial activation of T cells with a norm of MA = from 152.7 to 175.9%, for a population of CD3 + / CD4 + (T-helpers) a decrease in MA below 155.6% indicates mitochondrial insufficiency of T-helpers, an increase in MA above 187.6% indicates mitochondrial activation of T-helpers with a norm of MA = from 155.6 to 187.6%, for a population of CD3 + / CD8 + (T-cytotoxic), a decrease in MA below 135.1% indicates mitochondrial insufficiency of T-cytotoxic lymphocytes, an increase in MA above 158.1% indicates mitochondrial activation of T-cytotoxic lymphocytes with a norm of MA = from 135.1 to 158.1%, for a population of CD3 + / CD19 + (B cells), a decrease in MA below 133.6% indicates mitochondrial B cell deficiency, an increase in MA above 157.4% indicates about mitochondrial activation of B cells with a norm of MA = from 133.6 to 157.4%, for a population of CD3-CD16 + CD56 + (NK cells) Reductions below 98.3% AI indicates mitochondrial insufficiency NK cells, increased AI above 158.3% indicates mitochondrial activate NK cells in normal AI = from 98.3 to 158.3%.

A significant difference between the proposed solution and the known prototype is that venous blood lymphoconcentrate treated with monoclonal antibodies with a fluorescent label is used for analysis, and flow laser cytometry is used as an instrumental method, and the mitochondrial activity index in the selected populations is determined by the formula:

MA = KBS1 / KBS2 · 100%, where

MA - mitochondrial activity,

KBS1 - the average coefficient of intensity of lateral light scattering in a population of cells before the cytochemical reaction,

KBS2 is the average coefficient of intensity of lateral light scattering in a population of cells after a cytochemical reaction.

The method is as follows.

1. Label tubes for each patient per two samples.

Test tubes of the first standard test (A, B, C, D, E) are intended for standard immunological reactions. Test tubes of the second sample "cytochemistry" (B +, C +, D +, E +) are intended for immunocytochemical reactions.

A - CD45 / CD14 (for identification of a population of leukocytes and allocation of a lymphocytic window by small-angle and lateral light scattering)

B - CD3 / CD19 (to determine the number of T and B lymphocytes, respectively)

C - CD3 / CD4 (for determining the number of T-helpers - CD3 + CD4 +)

D - CD3 / CD8 (for determining the number of cytotoxic T-lymphocytes -CD3 + CD8 +)

E - CD3 / CD16, CD56 (for determining the content of natural killers

NK-CD3-CD16 + CD56 +)

B + - CD3 / CD19 (to determine the activity of LDH in T- and B-lymphocytes, respectively)

C + - CD3 / CD4 (for determining the activity of LDH in T-helper CD3 + CD4 +)

D + - CD3 / CD8 (for determining the activity of LDH in cytotoxic T-lymphocytes - CD3 + CD8 +)

E + - CD3 / CD16, CD56 (for determining the activity of LDH in natural killers NK - CD3-CD16 + CD56 +).

Pipette 4 µl of antibodies corresponding to the labeling of the tube into each tube with a clean tip.

2. Isolation of a mononuclear suspension.

3 ml of the patient’s venous blood is taken into a sterile tube with EDTA (BD Vacutainer No. 36860).

A blood sample is diluted in a 1: 3 ratio with RPMI-1640 medium (PanEco software), preheated to 37 ° C. Diluted blood is layered on a Ficoll-Hypac gradient with a density of 1.077 (Histopaque-1077 Sigma diagnostics No. 1077-1) in a 4: 1 ratio in a siliconized tube with a conical bottom. The sample is centrifuged for 20 minutes at 1850 rpm.

After centrifugation with an pipette, an interphase ring containing mononuclear cells is collected. After that, the cells are washed with Cell Wash cell reagent reagent (BD Cell Wash No. 349524) (5 minutes at 1500 rpm). The supernatant was removed, Cell Wash reagent was added to the sediment to a total volume of 450 μl.

3. The resulting cell suspension is added to labeled tubes of 50 μl each.

The tubes are kept in the dark at room temperature for 15 minutes.

4. The cell suspension of the standard sample (tubes A, B, C, D, E) is washed with Cell Wash reagent (5 minutes at 1500 rpm). After washing, 200 μl of CellFIX fixing solution (BD CellFIX No. 340181) is added to each cell suspension in a test tube, and the samples are ready for analysis on a flow cytometer.

5. Cells of the cytochemistry sample (B +, C +, D +, E + tubes) are permeabilized for 20 minutes at room temperature with Cytofix / Cytoperm reagent (BD Cytofix / Cytoperm No. 554722), after which the cells are washed with Cell Wash reagent (5 minutes at 1500 rpm). Then the cells are kept in Perm / Wash reagent (BD Pharmingen Perm / wash Buffer No. 554723) for 15 minutes and washed with Cell Wash reagent (5 minutes at 1500 rpm).

At the next stage, 1 ml of incubation medium is added to the cells of the “cytochemistry” sample (B +, C +, D +, E + tubes) to determine the activity of LDH, the cells are incubated for 30 minutes in an aqueous thermostat at a temperature of 37 ° C. After that, the cells are washed with Cell Wash reagent (5 minutes at 1500 rpm). After washing, 200 μl of Cell Wash reagent is added to each cell suspension to each tube; samples are ready for analysis on a flow cytometer.

The indicators obtained by cytofluometry are entered in the table.

The following are clinical examples.

Example 1

Patient P. 7 years. (Examination during medical examination)

Dz: healthy.

Sample "standard" cell type cell percentage KBS environments AT CD19 + (B lymphocytes) 9 144.51 CD3 + (T-lymphocytes) 63 154.73 FROM CD3 + CD4 + (T-helpers) 31 144.2 CD3 + (T-lymphocytes) 63 157.49 D CD3 + CD8 + (T-cytotoxic) 27 156.72 CD3 + (T-lymphocytes) 63 147.67 E CD3-CD16 + CD56 + (NK cells) 26 159.34 CD3 + (T-lymphocytes) 63 147.82 Sample "cytochemistry" cell type cell percentage KBS environments B + CD19 + (B lymphocytes) 9 198.99 CD3 + (T-lymphocytes) 63 290.09 C + CD3 + CD4 + (T-helpers) 31 252.45 CD3 + (T-lymphocytes) 63 327.01 D + CD3 + CD8 + (T-cytotoxic) 27 266.03 CD3 + (T-lymphocytes) 63 294.91 E + CD3-CD16 + CD56 + (NK cells) 26 249.83 CD3 + (T-lymphocytes) 63 285.21

Based on the protocol data, a resulting table is compiled and growth values are calculated for each population; for the CD3 + population (T-lymphocytes), data are averaged over 4 samples.

Indicator % cells Average lateral light scattering coefficient Growth Percentage (%) = MA "standard" "cytochemistry" CD3 + (T-lymphocytes) 63 151.93 299.31 197.00 CD19 + (B lymphocytes) 9 144.51 198.99 137.99 CD3 + CD4 + (T-helpers) 31 144.2 252.45 175.07 CD3 + CD8 + (T-cytotoxic) 27 156.72 266.03 169.75 CD3-CD16 + CD56 + (NK cells) 26 159.34 249.83 156.79

Conclusion:

Mitochondrial activation of T cells due to moderate mitochondrial activation of a population of T-cytotoxic lymphocytes. The child has moderate metabolic disturbances that require correction with a short course of metabolite therapy.

Example 2. Patient D. 11 years

Clinical diagnosis: Atopic dermatitis, common form, severe course. Erythroderma. Staphyloderma. Bronchial asthma, atopic form, moderate to severe course. Food allergy. Year-round allergic rhinitis. Chronic gastroduodenitis.

Sample "standard" cell type cell percentage KBS environments AT CD19 + (B lymphocytes) fourteen 146.81 CD3 + (T-lymphocytes) 76 155.1 FROM CD3 + CD4 + (T-helpers) 53 153.55 CD3 + (T-lymphocytes) 76 156.52 D CD3 + CD8 + (T-cytotoxic) 19 155.49 CD3 + (T-lymphocytes) 75 151.04 E CD3-CD16 + CD56 + (NK cells) 2 175.74 CD3 + (T-lymphocytes) 80 180.29 Sample "cytochemistry" cell type cell percentage KBS environments B + CD19 + (B lymphocytes) fourteen 213.49 CD3 + (T-lymphocytes) 78 274.83 C + CD3 + CD4 + (T-helpers) 53 266.53 CD3 + (T-lymphocytes) 78 276.14 D + CD3 + CD8 + (T-cytotoxic) 19 348 CD3 + (T-lymphocytes) 75 297.21 E + CD3-CD16 + CD56 + (NK cells) 2 404.11 CD3 + (T-lymphocytes) 80 279.33

Indicator % cells Average lateral light scattering coefficient Growth Percentage (%) "standard" "cytochemistry" CD3 + (T-lymphocytes) 77 155.74 281.83 180.96 CD19 + fourteen 146.81 213.49 145.42 (B-lymphocytes) CD3 + CD4 + (T-helpers) 53 153.55 266.53 173.58 CD3 + CD8 + (T-cytotoxic) 19 155.49 348 223.81 CD3-CD16 + CD56 + (NK cells) 2 175.74 404.11 229.95

Conclusion:

Pronounced mitochondrial activation of T-cytotoxic lymphocytes and NK cells with a sharp decrease in the number of these cells, which indicates a significant functional activation of cells in these populations against the background of a decrease in their number. The child has significant immunometabolic disorders that require correction by both metabolic drugs and immunomodulators.

Thus, the invention made it possible to measure mitochondrial activity in individual lymphocyte populations. The use of lymphoconcentrate, and not lymphocytes fixed on glass, allowed to reduce the amount of substrate of the enzyme reaction by 20 times, the incubation time of cells by 2 times compared with the prototype. To evaluate the result of the cytochemical reaction, the most modern method of cytological research is used - laser flow cytometry, which allowed us to evaluate the result of the cytochemical reaction in a larger number of cells (in the prototype, 50 cells are analyzed, in the present solution up to 5000 cells, which increases the reliability of the study. In addition, differentiated assessment of the result of the cytochemical reaction in individual cell populations, which is achieved by the possibility of immunophenotyping and tracked cells.

Such a change in technical parameters allows simultaneous immunological and cytochemical reactions in a venous blood sample, which leads to a more detailed assessment of both immunological and metabolic changes in the body and increases the diagnostic significance of the analysis.

Claims (1)

A method for measuring mitochondrial activity of lymphocytes, which includes measuring the activity of succinate dehydrogenase in lymphocytes by incubating permeabilized cells in an incubation medium consisting of a substrate specific for a given enzyme - succinic acid, phosphate buffer, n-nitrotetrazolium violet and trilon B, characterized in that for analysis, a venous blood lymphoconcentrate treated with monoclonal antibodies with a fluorescent label is used to identify various populations lymphocytes, moreover, flow laser cytometry is used as an instrumental method, while the indicator of mitochondrial activity in the selected populations is determined by the formula MA = KBS1 / KBS2 · 100%, where MA is mitochondrial activity, KBS1 - the average coefficient of intensity of lateral light scattering in a population of cells before the cytochemical reaction, KBS2 - the average coefficient of intensity of lateral light scattering in a population of cells after a cytochemical reaction, at the same time, for a population of CD3 + (T cells), a decrease in MA below 152.7% indicates mitochondrial T cell deficiency, an increase in MA above 175.9% indicates mitochondrial activation of T cells, with a norm of MA = 152.7 to 175 , 9%, for the population of CD3 + / CD4 + (T-helpers), a decrease in MA below 155.6% indicates mitochondrial insufficiency of T-helpers, an increase in MA above 187.6% indicates mitochondrial activation of T-helpers, with a norm of MA = from 155 , 6 to 187.6%, for a population of CD3 + / CD8 + (T-cytotoxic), a decrease in MA below 135.1% indicates mitochondrial underdevelopment T-cytotoxic lymphocyte deficiency, an increase in MA above 158.1% indicates mitochondrial activation of T-cytotoxic lymphocytes, with a norm of MA = 135.1 to 158.1%, for a population of CD19 + (B cells), a decrease in MA is below 133.6 % indicates mitochondrial B cell deficiency, an increase in MA above 157.4% indicates mitochondrial activation of B cells, with a norm of MA = 133.6 to 157.4%, for a population of CD3-CD16 + CD56 + (NK cells) a decrease in MA below 98.3% indicates mitochondrial NK cell deficiency, an increase in MA above 158.3% indicates mitochon dry activation of NK cells, with a norm of MA = from 98.3 to 158.3%.

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