RU2495423C1 - Method for lymphocyte functional activity assay in chronical adenoiditis in children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves the unified assay of quality and quantity of the correlation of laboratory parameters with calculating a significant value of lymphocytic enzyme functional activity. If the significant value of lymphocytic enzyme activity (K) is 0.9 or more, lymphocyte activity is considered to be adequate and pre-determining the favourable clinical course of chronic adenoiditis, while the significant value of lymphocytic enzyme activity is less than 0.9, lymphocyte hypoactivity pre-determining the unfavourable clinical course of chronic adenoiditis is stated.

EFFECT: reliable, informative, time-optimised the functional activity assay of lymphocyte populations, reducing the time of diagnosis, analysis simplicity.

3 dwg, 3 ex

Description

The invention relates to medicine, namely to otorhinolaryngology, and can be used to determine the functional activity of lymphocytes in children with chronic adenoiditis, as well as to assess the severity and course of the disease.

The closest technical solution is a cytogenetic method for assessing the functional activity of blood T-lymphocytes of a patient, including their isolation from a venous blood sample, which is then cultured for 48 hours on a nutrient medium with the addition of phytohemagglutinin, after which colchicine is added to a final concentration of 0.5 μg / ml and further cultured T-lymphocytes for 2 hours. The resulting culture fluid is centrifuged, the supernatant is treated with a hypotonic solution and centrifuged again. The obtained lymphocytes are fixed and preparations are prepared from them on glass slides. Count the number of lymphocytes containing at least five acrocentrics (CL5) in one acrocentric chromosome association (AAX), and the number of lymphocytes not containing AAX (CL0). The percentage of these lymphocytes from the total number of lymphocytes in metaphase is determined. With an increase in the percentage of CL0 by no less than 20% compared with the value of the indicated indicator obtained from this patient earlier, and the presence of CL5, the activity of T-lymphocytes is assessed as adequate. With an increase in the percentage of CL0 by at least 60% compared to the value of the indicated indicator obtained from this patient earlier, and in the absence of CL5 or with a decrease in the percentage of CL0 by at least 60% and an increase in the percentage of CL5 by more than two times compared with the values of these indicators obtained from this patient earlier, the activity of T-lymphocytes is evaluated as reduced (RF Patent No. 2112984, M. cl. G01N 33/49, C12N 5/08 1995).

The disadvantage of this method is to determine the activity of part of the blood lymphocytes, namely T-lymphocytes, which act on antigen-loaded target cells. The limitation in the assessment of the functional activity of all lymphocytes was predetermined by the use of phytohemagglutinin, since T-lymphocytes that determine the cellular immune response of the body respond to it. Assessment of the level of acrocentric chromosome association in lymphocytes does not indicate the functional activity of all lymphocytes: B-lymphocytes, natural killer cells, which are activated during delayed hypersensitivity reactions. To assess the functional activity of lymphocytes, different populations of lymphocytes are of great importance, in the differentiation of which obligatory somatic recombination of deoxyribonucleic acid and segments of receptor genes for antigens takes place, forming a total immune response, on which an early prospective determination of the course of the disease depends.

In the known method, the disadvantages include the additional use of phytohemagglutinin as a stimulator of the proliferative activity of T-lymphocytes in addition to the etiological antigenic stimulus in the disease, which introduces an excess stimulus and creates an additional background of lymphocyte activity, forming a measurement error. The known method does not take into account the basal level of functional activity and proliferation of viable lymphocytes, which is observed and maintained throughout the life of the body, and also provides recognition of the cells of your body. This functional activity and proliferation differs from the expansion of a clone of lymphocytes in the process of developing an active, productive immune response to an antigen to be eliminated from the body, and is not associated with the synthesis and secretion of active T-lymphocyte cytokines and the induction of immune inflammation.

The disadvantage of this method is the lack of microscopic control of the morphological composition of lymphocytes. With a similar structure of the structures of the nucleus of lymphocytes and other leukocyte suspensions - monocytes, blast hematopoietic cells, granulocytes - chromosome preparations do not exclude the analysis of associations of acrocentric chromosomes of other leukocyte suspensions in the studied sample, characterizing the activity of lymphocytes.

The disadvantages of this method include the study of non-viable lymphocytes. Three changes in the fixation of T-lymphocytes on glass and the long-term implementation of the method for more than 48 hours preclude the determination of the activity of viable lymphocytes in real time. Evaluation of the functional activity of lymphocytes inherent in viable cells, when ascertaining their non-viability, is unconvincing.

The disadvantages of this method include the use for evaluating the functional activity of T-lymphocytes microscopic analysis of AAX with standard light microscopic testing of drugs in 100-200 cells from each individual. Subjective visualization without automated determination reduces the objectivity of the method.

The disadvantage of this method is to evaluate the activity of T-lymphocytes in case of complications in patients retrospectively, which does not allow prospectively determine the course and prognosis of the outcome of the disease in the early stages. This is demonstrated in the examples for various diseases.

In the known method, the disadvantages include the assessment of the functional activity of T-lymphocytes in the examined patients with a wide range of diseases, which does not characterize specific pathogenetic reactions exclusively for a specific nosology. Changes in the functional activity of T-lymphocytes of a general nature do not allow determining the course options for a specific disease.

The disadvantage of this method is the duration of the cytogenetic assessment of more than 48 hours, which delays the diagnosis of acute or exacerbation of a chronic disease with a severe course. The use of a venous blood sample in an amount of 5.0 ml and repeated taking exacerbates the invasiveness of the method.

The objective of the invention is the creation of a reliable informative and time-optimized method for early prospective determination of the nature of the course of chronic adenoiditis in children, due to the functional activity of lymphocytes, which determines the differentiated tactics of managing sick children.

The problem is achieved in that in a method for determining the functional activity of lymphocytes in children with chronic adenoiditis, including the separation of lymphocytes from venous blood by centrifugation, treatment of the supernatant with sodium chloride solution, centrifugation of diluted blood and counting the number of lymphocytes, the new is that they determine the purity of lymphocyte output by microscopic control of the morphological composition of leukocyte suspensions, then determine the viability of lymphocytes stained with a trypan blue solution, osmotic lysis of a suspension of isolated lymphocytes containing cells at a concentration of 1.0 million / ml is carried out by introducing distilled water 1: 5 by volume into a suspension of lymphocytes, 1.0-2.0 ml of dithiothreitol, the resulting mixture of destroyed lymphocytes in 200 μl are incubated with 600 μl of a substrate with a concentration of 0.50-8.70 mm and a cofactor with a concentration of 0.50-8.10 mm and determine the preliminary relative value of the functional activity of lymphocyte enzymes by measuring the glow when mixing incubation cm B and bioluminescent reagents, the absolute value of the functional activity of the lymphocytes is determined by enzyme calibration curve of fluorescence intensity versus concentration of lymphocytes enzymes and functional activity was determined according to the formula:

; $$A=\frac{\Delta C\times V\times \mathrm{one}{0}^{-6}}{T}$$

;

Where

A - functional activity of lymphocyte enzymes, E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

ΔС is the difference in the concentrations of lymphocyte enzymes in absolute and relative values, mmol;

V is the sample volume, in milliliters;

T is the incubation time, in minutes;

the value of a significant indicator of the activity of lymphocyte enzymes is determined by the formula:

; $$K=\frac{A_{\mathrm{one}}+A_2}{A_3+A_{\mathrm{four}}}$$

;

Where

K is a significant indicator of the activity of lymphocyte enzymes;

And ₁ - the functional activity of the enzyme lactate dehydrogenase (LDH) lymphocytes, E (unit activity of the enzyme) for 10 ⁴ lymphocytes (1E = 1 μmol / min);

A ₂ - functional activity of the enzyme nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADPHDG), E (unit activity of the enzyme) for 10 ⁴ lymphocytes (1E = 1 μmol / min);

A ₃ - functional activity of the enzyme malate dehydrogenase (MDH), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

A ₄ - functional activity of the enzyme nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase (NADGDG), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

when the value of a significant indicator of the activity of lymphocyte enzymes (K) is equal to or more than 0.9, the activity of lymphocytes is determined as an adequate, predetermining a favorable course of chronic adenoiditis, and when the value of a significant indicator of the activity of enzymes of lymphocytes (K) is less than 0.9, the activity of lymphocytes is determined as a reduced, determining the unfavorable course of chronic adenoiditis.

Chronic adenoiditis (HA) is a polyetiological disease, which is based on a violation of the physiological immune processes of the pharyngeal tonsil. The immune system, having local representations in the organs, is one in the body and ensures its viability.

In the proposed method, the functional activity of all populations of lymphocytes is determined. Lymphocytes represent the morphological basis of the immune system. The interaction of all populations of lymphocytes forms the body's immune response initiated by the pathogen. The integration of lymphocytes in the immune response provides pre-immune inflammation, recognition of damaged cells, destruction of damaged tissues during the immune inflammatory process, removal of degradation products from the body. A precise determination of the activity of all lymphocytes establishes the immune mechanisms involved in the disease, which determines the course and the correction of the tactics of managing sick children.

In the proposed method, the only etiological antigenic stimulus of chronic adenoiditis acts as a stimulator of proliferative activity (phase of increasing immune response) of lymphocytes. Lymphocytes (both T-lymphocytes and B-lymphocytes) carry specific antigenic cell receptors, the phenotype of which is regulated by external activation conditions. They begin proliferation after meeting with the corresponding antigen, specifically binding it to membrane high affinity receptors, forming clones of specific effector cells. Plasma cells secreting immunoglobulins (antibodies) are formed from B-lymphocytes, and cytotoxic cells from T-lymphocytes that suppress pathogenetic reactions in chronic adenoiditis. The natural model of in vivo proliferation of lymphocytes is not the same as the proliferation of B-, T-lymphocytes in vitro, and it plays a central role, allowing one to reliably characterize intracellular processes and induction of the intracellular signal to proliferation.

In the proposed method, the purity of the output of lymphocytes is determined by conducting microscopic control of the morphological composition of leukocyte suspensions. Morphological identification of the studied lymphocytes in the amount of 1 million selected cells per milliliter with the control of exclusion of other leukocyte suspensions are objective criteria for reliable determination of functional activity and exclude false-positive results.

In the proposed method, the viability of lymphocytes is determined by staining with a solution of trypan blue. This dye does not penetrate the membranes of living cells, but when damaged, it is able to stain the cell nucleus. Counting of lymphocytes is carried out in the Goryaev’s chamber. The proportion of viable lymphocytes, comprising at least 97% in the sample, objectively characterizes their functional activity and reflects it in real time. Monitoring the viability of lymphocytes eliminates methodological inaccuracies in the formulation of reactions.

In the proposed method, osmotic lysis of a suspension of isolated lymphocytes containing cells at a concentration of 1.0 million / ml is carried out by introducing distilled water of 1: 5 by volume and 1.0-2.0 ml of dithiothreitol into the lymphocyte suspension, which creates the conditions for high-quality preparation samples of biological material. Firstly, this ensures complete lysis of all lymphocytes in 100%, since the osmotic potential of the lymphocyte is greater than the osmotic potential of distilled water. Secondly, the use of dithiothreitol as a modifier protects the SH-groups of the protein components of the cell from uncontrolled oxidation during the lysis and prevents the formation of abnormal intramolecular and intermolecular-S-S-bonds, which is important for the subsequent determination of enzyme activity. Thirdly, when dithiothreitol is bound to the membrane of lymphocytes, the interaction of the cytoskeleton with membrane proteins is weakened, in which the shape of the white molecules and the tendency to develop lysis do not change.

Evaluation of the traditional immune status in chronic adenoiditis with a calculation of the population and subpopulation composition of blood lymphocytes does not give an unambiguous answer about the presence and / or severity of immunodeficiency.

Changes in the quantitative composition of lymphocytes occur with severe breakdowns on the part of the immune system. At the same time, early and / or qualitative signs of disorders alter the functional activity of the cell, forming a response to external influences, which allows us to gain an idea of the metabolic strategy of the immune response chosen by the body. The functional activity of the blood lymphocyte reflects the state of metabolic and regulatory processes of the body: catabolism, synthesis, utilization, aerobic and anaerobic respiration. The informational content of metabolic reactions in lymphocytes allows the use of enzyme activity as an indicator of their activity. Enzymes that most objectively reflect the main parameters of intracellular exchange include enzymes of the oxidoreductase class, which catalyze the oxidation of one substance (electron and proton donor), coupled with the reduction of another compound (their acceptor): nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent (NAD and NAD -) dehydrogenases in blood lymphocytes.

In the proposed method, the resulting mixture of destroyed lymphocytes in an amount of 200 μl is incubated with 600 μl of substrate and cofactor with specific concentrations, as well as the pH of the medium for each enzyme to be determined, which confirms the sensitivity, specificity, and information content of the method.

The choice as criteria of indicators of activity of the enzymes LDH, MDH, NADGDG, NADPHDG is determined by the results of determining the activity of NAD- and NADP-dependent enzymes in blood lymphocytes in children with chronic adenoiditis, significantly differing from the corresponding activity indicators of lymphocyte enzymes in healthy children (Fig. 1).

The activities of NAD (F) -dependent dehydrogenases of blood lymphocytes in children with CA reflect the characteristic metabolic mechanisms of the pathological process: insufficient energy processes that provide the basic needs of cells in adenosine triphosphoric acid (ATP). The lymphocyte metabolism in CA is characterized by a low level of anaerobic and aerobic respiration and a decrease in the substrate interaction of the tricarboxylic acid cycle with amino acid metabolism reactions. The mechanism causes an increase in the level of plastic metabolic reactions and the ability of cells to proliferate, which is manifested in chronic adenoiditis with hypertrophy of lymphadenoid tissue.

LDH is a glycolysis enzyme that reversibly catalyzes the oxidation of lactate to pyruvic acid with the participation of NAD as a coenzyme. LDH occupies a key position in the regulation of the cytoplasmic level of NADI / NAD. In the case of an excess of NADH in the cytoplasm, LDH reduces pyruvate to lactate, which is then removed from the cell (anaerobic LDH reaction). When aerobic processes are activated, LDH can oxidize lactate to pyruvate to form NADH (aerobic LDH reaction).

MLH, one of the key enzymes of the Krebs cycle, catalyzes the reversible oxidation of malate to oxaloacetate. The enzyme is localized both in mitochondria (the tricarboxylic acid cycle) and in the cytoplasm. In mitochondria, the level of the NADH / NAD ratio is relatively high, as a result of which the intramitochondrial oxaloacetate is reduced to manat, which easily leaves the mitochondria. In the cytoplasm, the level of the NADH / NAD ratio is small, which leads to the oxidation of malate with the participation of the cytoplasmic NAD-dependent MDH.

Glutamate dehydrogenases carry out oxidative deamination of G-glutamic acid. Two glutamate dehydrogenases are distinguished, using NAD or NADP as cofactors. The enzymatic reactions of glutamate dehydrogenases are reversible, respectively, ammonia in the presence of NAD (F) H and α-ketoglutaric acid can participate in the synthesis of glutamate. NAD- and NADP-dependent glutamate dehydrogenases are regulatory in the amino acid metabolism system. NADGDG characterizes the level of substrate (the breakdown products of amino acids) in the tricarboxylic acid cycle.

In the proposed method, a preliminary relative value of the functional activity of lymphocyte enzymes is determined by measuring the glow when mixing the incubation mixture and bioluminescent reagents, which eliminates preanalytical errors in determining the functional activity of lymphocyte enzymes. In laboratory determination and mathematical calculations of the preliminary relative value of the functional activity of lymphocyte enzymes, the indicators conventionally called "substrate background of enzymes" were taken into account. These indicators are due to the presence in the cells of a certain amount of substrates for the course of various metabolic reactions catalyzed by the studied enzymes. Activated lymphocytes are powerful effectors that trigger mechanisms of cascade reactions, providing the development of inflammation. The anti-infective effect of lymphocytes is associated with the recombination of free radicals and enzymatic oxidation reactions. The main activators of free radical recombination and enzymatic oxidation are bioluminescent reagents bacterial luciferase from Photobacterium leiognathi and oxidoreductase from Vibrio fischeri. Carrying out the reaction in viable lymphocytes with the priority of the release and fixation of energy in the light form compared with the heat is a simple informative method that allows you to objectively evaluate the oxygen-dependent biocidality of lymphocytes.

In the proposed method, the absolute value of the functional activity of lymphocyte enzymes is determined according to the calibration graph of the dependence of the luminous intensity on the concentration of lymphocyte enzymes, which ensures the conclusions are correct, high sensitivity and specificity, which dictate the liquidity of the method. The sensitivity and specificity of the method emphasize significant changes in the activity of lymphocyte enzymes in children, characteristic of chronic adenoiditis, in contrast to similar indicators in other inflammatory diseases.

In the proposed method, the functional activity of lymphocytes is determined automatically, with the assessment of the results, regardless of subjective factors, using the presented formula in generally accepted units of measurement, which is expressed mathematically quantitatively and excludes the subjectivity of a qualitative assessment.

In the proposed method, the value of a significant indicator of the activity of lymphocyte enzymes (K) is determined as a new reliable differentiated criterion - the coefficient of plastic and energy metabolism integratively characterizing the level of intensity of plastic and energy processes. When the coefficient value is equal to or more than 0.9, the prevalence of plastic processes in lymphocytes is noted (NADPH production increases), the activity of lymphocyte enzymes is determined as adequate, on the basis of which the favorable course of chronic adenoiditis is predetermined. When the coefficient value is less than 0.9, the prevalence of energy processes in lymphocytes, the reduction of macroglobular synthesis reactions (including the synthesis of cell receptors), the activity of lymphocyte enzymes are determined as reduced, on the basis of which the unfavorable course of chronic adenoiditis is determined.

The value of 0.9 was obtained empirically based on a comparison of the calculated coefficient values, clinical observation data of children suffering from chronic adenoiditis. The value of a significant indicator of the activity of lymphocyte enzymes is a dimensionless value that does not depend on the sensitivity of the bioluminometer on which the study is conducted.

In the proposed method, reproducibility is positive under the conditions of a standard clinical and biochemical laboratory that does not require equipment for working with cell culture, low cost, reduced diagnostic time to 2 hours, which allows it to be widely used, optimizing the determination of the functional activity of lymphocytes in chronic adenoiditis. The use of a small volume of a venous blood sample in an amount of 2.0 ml and a single dose reduces the invasiveness of the method.

The method is as follows.

A patient with chronic adenoiditis in the morning, on an empty stomach, take a peripheral blood sample from the ulnar vein in a volume of 2.0 ml into a vacuum tube with a preservative containing Li-heparin as an anticoagulant. The study is carried out within one hour after taking the blood. Lymphocytes are isolated from venous blood according to the method proposed by A. Boyum (1968) by centrifugation in a density gradient of ficol-verographin (ρ = 1,077 g / cm ³ ), dividing blood cells into:

a) the mononuclear fraction (MF), which includes lymphocytes, monocytes and blast hematopoietic cells;

b) a fraction containing granulocytes and red blood cells.

For this, venous blood is mixed with a solution of sodium chloride 0.9% (pH 7.2) in a ratio of 1: 2-1: 3. Enriched with lymphocytes plasma is obtained by centrifugation of stabilized blood at 400 g for 30 minutes in a density gradient of ficol-verographin (ρ = 1,077 g / cm ³ ).

The resulting mononuclear fraction (MF) is washed 3 times in a solution that contains 0.9% sodium chloride solution, buffered with phosphate-buffered saline (PBS). Resuspendable in nutrient medium RPMI-1640 (Roswell Park Memorial Institute medium, Eng.) In the amount of 10 ⁶ cells in 1 ml. Microscopic control of the morphological composition of leukocyte suspensions is carried out by counting the number of lymphocytes in the Goryaev chamber to determine the purity of the output of lymphocytes, which should be at least 97%. Lymphocyte viability is then determined by staining with a trypan blue solution.

Destroy the lymphocytes by osmotic lysis by adding to the suspension of lymphocytes containing cells at a concentration of 1.0 million / ml, distilled water (1: 5 by volume) and 1.0-2.0 mm dithiothreitol.

The activity of lymphocyte enzymes - LDH, NADPHDG, MDH and NADGDG - is determined using the bioluminescent method. To do this, in 600 μl of the incubation mixture containing the appropriate substrate and cofactor, add 200 μl of a suspension of destroyed lymphocyte cells. Specific values of the concentrations of substrates and cofactors, as well as the pH of the medium for the determination of enzymes are presented in figure 2.

In an incubation mixture for determining the activity of NADH-dependent reactions NADGDG and NADPH-dependent reactions NADPHDG additionally add NH ₄ Cl at a concentration of 5.0 mm.

After incubation at 37 ° C for 30 minutes (for enzymatic reactions with reduction of NAD (F)) or 5 minutes (for the reaction with oxidation of NAD (F) H), 50 μl of flavin mononucleotide (FMN) in a concentration of 1.5 is added to 200 μl of the incubation mixture × 10 ^-5 M, 10 μl of the NAD (F) N enzyme system: FMN oxidoreductase-luciferase and 50 μl of 0.0005% myristic aldehyde C ₁₄ (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ - phosphate buffer with pH 7.0). The NAD (F) N enzymatic system: FMN oxidoreductase-luciferase is made from luciferase purified from ion-exchange chromatography and gel filtration from Photobacterium leiognathi and oxidoreductase from Vibrio fischeri at the Institute of Biophysics SB RAS. After mixing the bioluminescent reagents and the incubation mixture using a biochemiluminometer, for example, brand "BLM-8803", measure the glow. Given that the cells have a certain number of substrates for the course of various metabolic reactions, including those catalyzed by the studied enzymes, the parameters conventionally called “substrate background of enzymes” are determined. The definition of “substrate enzyme backgrounds” is carried out under the same conditions as for the above dehydrogenases, but a buffer is added to the incubation mixture instead of the corresponding substrate. As a result of measuring the glow on a biochemiluminometer, a preliminary relative value of the activity of the studied enzymes is obtained. To obtain the absolute value of the activity of enzymes (conventionally called "enzyme"), graphs are constructed of the dependence of the intensity of bioluminescence on the concentration of NAD (F) H (calibration graph). For this, 200 μl of a standard solution of NAD (F) N in the range of 10 ^-9 -10 ^-4 M is added to the bioluminometer cuvette containing FMN, NAD (F) N: FMN oxidoreductase-luciferase and myristin aldehyde in the concentrations indicated above, and then carried out measurement of bioluminescence intensity. Due to the wide range of pH buffers used to determine dehydrogenase activity, as well as the pH dependence of the bioluminescence of the enzymatic system from luminous bacteria, calibration plots are plotted for each pH buffer. The functional activity of lymphocyte enzymes (or NAD (F) N-dehydrogenases) is determined by the formula:

; $$A=\frac{\Delta C\times V\times \mathrm{one}{0}^{-6}}{T}$$

;

Where

A - functional activity of lymphocyte enzymes, E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

ΔC is the difference in the concentrations of lymphocyte enzymes in absolute and relative values ("enzyme" and "substrate background of the enzyme"), mmol;

V is the sample volume, in milliliters;

T is the incubation time, in minutes.

Then determine the value of a significant indicator of the functional activity of lymphocyte enzymes according to the formula:

; $$K=\frac{A_{\mathrm{one}}+A_2}{{\mathrm{<figure-callout\; id="3"\; label="A"\; filenames="00000006.png,00000008.png"\; state="\{\{state\}\}">A</figure-callout>}}_3+A_{\mathrm{four}}}$$

;

Where

K is a significant indicator of the activity of lymphocyte enzymes,

A ₁ - functional activity of the enzyme lactate dehydrogenase (LDH) lymphocytes, E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

A ₂ - functional activity of the enzyme nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADPHDG), E (unit activity of the enzyme) for 10 ⁴ lymphocytes (1E = 1 μmol / min);

A ₃ - functional activity of the enzyme malate dehydrogenase (MDH), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);

And ₄ is the functional activity of the enzyme nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase (NADGDG), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min).

When the value of a significant indicator of the activity of lymphocyte enzymes (K) is equal to or more than 0.9, the activity of lymphocytes is determined as an adequate, predetermining favorable course of chronic adenoiditis. When the value of a significant indicator of the activity of lymphocyte enzymes is less than 0.9, the activity of lymphocytes is determined as a reduced, predetermining unfavorable course of chronic adenoiditis.

This method is confirmed by the results of a dynamic examination of 56 children suffering from chronic adenoiditis. Upon initial treatment, all children with chronic adenoiditis underwent a commission endoscopic ENT examination, an examination by an allergist and pediatrician in combination with bacteriological, morphological, radiological (if indicated) and laboratory research methods. Laboratory methods included a detailed blood test, a blood test for immune status.

Additionally, a retrospective analysis of the coincidence of determining the variant course of chronic adenoiditis in 56 children was carried out. The coincidence of the determination of the variant course of chronic adenoiditis was ascertained by the fact of documented exacerbation of the disease after 90 days from the initial treatment in accordance with a lymphocyte lifespan of 30 to 90 days and comparison with a prospective determination of the functional activity of lymphocytes. The results are presented in figure 3.

According to the results of the otorhinolaryngological examination, by 90 days after the initial treatment, out of 24 children with chronic adenoiditis with adequate functional activity of lymphocytes and K≥0.9 in 23 children the symptoms of chronic adenoiditis were completely stopped, the number of exacerbations during the observation period was 1. Of 32 children with chronic adenoiditis with low functional activity of lymphocytes and K <0.9 in 30 children, the symptoms of the disease decreased, but were not stopped, the amount of exacerbation during the observation period Ia was 3. Total coincidence determining variant of chronic adenoiditis in children with determination of the meaningful indicator of enzyme activity was 94.5% (53 individuals).

Thus, a reliable determination of the course of chronic adenoiditis in children has been demonstrated, carried out by prospectively determining the functional activity of blood lymphocytes and the value of a significant indicator of the functional activity of lymphocyte enzymes.

The proposed method is illustrated by drawings, where:

Figure 1 shows

The activity of NAD (F) -dependent dehydrogenases in blood lymphocytes in healthy children and patients with chronic adenoiditis (Me, P ₂₅ -P ₇₅ ).

Figure 2 shows

The concentration of substrates, cofactors and pH indicators for determining the activity of NAD (F) -dependent dehydrogenases in blood lymphocytes by a bioluminescent method.

Figure 3 shows

The coincidence of the retrospective and prospective determination of the variant course of chronic adenoiditis by the value of a significant indicator of the activity of lymphocyte enzymes.

An example of the method

Example 1

Patient D., age 6 years 2 months (study protocol No. 26), applied for admission to the University Clinic of the Krasnoyarsk State Medical University of Krasnoyarsk on December 14, 2011. During the initial examination, she complained of mild difficulty in nasal breathing due to periodic congestion nose, frequent acute respiratory viral infections (SARS) 1 time per month, moderate loss of appetite, fatigue, general weakness, restless sleep with nightly fears. Complaints of a local and general nature within 2 years.

Associates complaints with the consequence of repeated SARS, an atypical course due to a prolonged period of the disease for more than 10 days, with a complication in the form of a recurring runny nose. During the year, 4 times verified exacerbation of chronic adenoiditis. She was repeatedly treated on an outpatient basis by a pediatrician and ENT specialist according to the generally accepted method, the last time two months ago: vasoconstrictor and sanitizing drops in the nose, endonasal lavage. Immunocorrection was not performed. As a result of the treatment, a short-term improvement of 10 days was noted due to a decrease in nasal congestion and a runny nose. The reason for applying for a second outpatient appointment was a long chronic progressive course of intoxication symptoms due to fatigue, loss of appetite, accompanied by a lack of complete relief of nasal congestion.

With endoscopy of the nasal cavity and nasopharynx, a mild swelling of the mucous membrane of the nasal cavity was noted in the region of the lower nasal concha with the phenomena of pathogenetically determined tissue and ventilation hypoxia and venous stasis in the form of clinically manifested cyanosis, moderate mucous discharge in the area of the hyperplastic pharyngeal tonsil of the II degree, making it difficult to outflow . On the part of other ENT organs, there were no signs of pathology. Lymph nodes of the posterior cervical group are enlarged to 0.7 × 0.7 cm in diameter, of a dense consistency, unsoldered, painless on palpation.

During the physical examination of patient D., periorbital, perioral, intermittent cyanosis was observed, intensifying during physical exertion, pallor of the face.

A clinical blood test did not reveal deviations in the number of red blood cells, hemoglobin, and leukocyte formula when a weakly accelerated erythrocyte sedimentation rate (ESR) of 11 mm / hour was found, which was a laboratory indicator of endogenous intoxication, indirectly reflecting the ratio in the blood plasma of protein particles of large sizes (globulins ) and small sizes (albumin) and testified to the inflammatory process.

The results of the study of the immune status of patient D. showed a significant increase of 12% in the relative number of all mature T-lymphocytes bearing a cluster of CD3 ⁺ cell differentiation (CD - Cluster of Differentiation, Eng.), In comparison with healthy children. The fact of a significant increase in the relative and absolute number of helper T-lymphocytes carrying CD3 ⁺ CD4 ⁺ , compared with healthy children, allowed us to state the absence of T-cell immunodeficiency. The revealed increase in the relative number of suppressor-cytotoxic T-lymphocytes carrying CD3 ⁺ CD8 ⁺ in patient D. with chronic adenoiditis characterized the period of the infectious process, when an increase in specific cytotoxic cells occurs. The values of the remaining indicators did not exceed the established limits of the norm for each of them.

During bacteriological examination of biological material from the surface of the pharyngeal tonsil, the reference value of the degree of contamination was revealed mainly due to the multiculture of opportunistic microflora - Staphylococcus aureus and staphylococcus saprophytic. The quantitative content of microorganisms per 1 g (ml) of the test material amounted to 1 × 10 ³ CFU / g (ml) (CFU - colony forming unit).

In the nasocytogram, patient D. showed a mild increase in neutrophils of 68.00% with a reference number of phagocytic neutrophils of 15.05%, which reflected the preserved phagocytic function of neutrophils.

According to the radiograph of the paranasal sinuses, no pathology was detected.

Taking into account complaints, anamnesis, objective examination data and a comprehensive clinical, laboratory and instrumental study by an otorhinolaryngologist, allergist and pediatrician, chronic adenoiditis, catarrhal form, remission were diagnosed.

A survey on the proposed method. In the peripheral blood lymphocytes of patient D., the relative and absolute values of the functional activity of lymphocyte enzymes were determined: A ₁ (LDH) = 51.54 μE per 10 ⁴ lymphocytes, A ₂ (NADPHDG) = 13.13 μE per 10 ⁴ lymphocytes, A ₃ (MDH) = 23.03 μE per 10 ⁴ lymphocytes, A ₄ (NADGDG) = 30.24 μE per 10 ⁴ lymphocytes. The value of a significant indicator of the functional activity of lymphocyte enzymes K = 1.2 was calculated and the favorable course of chronic adenoiditis was predetermined.

At the visit of patient D., 90 days after the initial treatment, a reliable objective clinical improvement was established. Complaints of a general nature did not bother, they feel well and have an emotional tone, the child is active. The appetite is satisfactory, the sleep returned to normal. With endoscopy of the nasal cavity and nasopharynx, the mucous membrane of the nasal cavity is pink, clean, swelling and mucous discharge in the area of the choan are stopped, providing free nasal breathing. Lymph nodes of the posterior cervical group 0.5 × 0.5 cm in diameter, elastic consistency, unsoldered, painless on palpation. ARVI did not tolerate reinfection, no exacerbation was observed. I did not receive drug immunocorrection.

In a clinical blood test, ESR was normalized, equal to 5 mm / hour, which indicated a favorable course of the disease and the patient's recovery.

Immunological examination showed that the studied immunological parameters did not exceed the established limits of the norm for each parameter.

In retrospect, the course of chronic adenoiditis has been identified as favorable with the elimination of immuno-metabolic disorders. The coincidence of the determination of the favorable variant of the course of chronic adenoiditis is complete - 100%.

Example 2

Patient X., age 4 years 6 months (study protocol No. 20), applied for admission to the University Clinic of the Krasnoyarsk State Medical University of Krasnoyarsk on November 10, 2011. Complaints during examination of mild difficulty in nasal breathing due to constant congestion and discharge from the nose, periodic morning cough, frequent SARS every month, a significant decrease in appetite with a delay in weight gain, fatigue, general weakness, restless sleep. Complaints of a local and general nature during the year. Associates complaints with admission to a kindergarten and repeated protracted ARVI. Repeated adenoiditis 4 times a year. Outpatiently received treatment at the pediatrician and ENT specialist according to the generally accepted method, the last time - 1 month ago: systemic antibacterial treatment of augmentin at an age dose, vasoconstrictor and sanitizing drops in the nose, endonasal lavage. Before treatment and during it, immunocorrection was not performed. The effectiveness of the treatment was insufficient, since it did not provide a complete long-term relief of symptoms of local and general nature. A periodic runny nose of a mucous character was preserved against the background of rapid fatigability, tearfulness, and delayed weight gain.

At endoscopy of the nasal cavity and nasopharynx, a moderate swelling of the mucous membrane of the nasal cavity in the region of the lower nasal concha was observed with the phenomena of pathogenetically caused tissue and ventilation hypoxia and venous stasis in the form of clinically manifested cyanosis, moderate mucous discharge with purulent component in the area of the cupularly located hyperplastic pharyngeal tonsil the degree that impedes its outflow, with the non-differentiation of the furrows. On the part of other ENT organs, there were no signs of pathology. Lymph nodes of the posterior cervical group are enlarged to 0.7 × 0.7 cm in diameter, of a dense consistency, unsoldered, painless on palpation.

In the clinical blood test, there were no deviations in the number of red blood cells, hemoglobin, leukocyte formula when a weakly accelerated erythrocyte sedimentation rate of 14 mm / hour was found, which was a laboratory indicator of endogenous intoxication.

The results of the study of the immune status of patient X. showed an unreliable increase in the relative number of all mature T-lymphocytes bearing a cluster of CD3 ⁺ cell differentiation by 5% compared with healthy children.

During bacteriological examination of biological material from the surface of the pharyngeal tonsil, the reference value of the degree of contamination was revealed mainly due to the multiculture of opportunistic microflora - Staphylococcus aureus and staphylococcus saprophytic. The quantitative content of microorganisms per 1 g (ml) of the test material amounted to 1 × 10 ⁴ KOE / g (ml).

Taking into account complaints, anamnesis, objective examination data and a comprehensive clinical, laboratory and instrumental study by an otorhinolaryngologist, allergist and pediatrician, chronic adenoiditis, purulent form, and remission were diagnosed.

A survey on the proposed method. The relative and absolute values of the functional activity of lymphocyte enzymes were determined in the patient’s peripheral blood lymphocytes: A ₁ (LDH) = 73.85 μE per 10 ⁴ lymphocytes, A ₂ (NADPHDG) = 0.91 μE per 10 ⁴ lymphocytes, A ₃ (MDG ) = 94.22 μE per 10 ⁴ lymphocytes, and ₄ (NADGDG) = 54.77 μE per 10 ⁴ lymphocytes. A significant indicator of the functional activity of lymphocyte enzymes K = 0.5 was calculated and an unfavorable course of chronic adenoiditis was predetermined.

At the visit of patient X. 90 days after the initial treatment, a reliable objective clinical improvement was established without relief of symptoms. General complaints decreased, but did not stop: health, emotional tone improved due to the manifestation of desires, the implementation of a sequence of actions. Appetite, sleep improved, but not normalized. With endoscopy of the nasal cavity and nasopharynx, the mucous membrane of the nasal cavity decreased cyanosis, swelling and mucous discharge in the choan region, providing improved nasal breathing. Lymph nodes of the posterior cervical group remained 0.7 × 0.7 cm in diameter, restoring the consistency to a tightly elastic one. SARS did not suffer reinfection. Exacerbations of chronic adenoiditis were noted 3 times, which required drug immunocorrection.

In the clinical blood test, a slightly accelerated erythrocyte sedimentation rate of 12 mm / h was preserved, indicating an unfavorable course of the disease.

A retrospective statement in patient X. of residual clinical signs of chronic adenoiditis of general and local nature confirmed the unfavorable course of the disease. The coincidence of the definition of an unfavorable variant of the course of chronic adenoiditis is complete - 100%.

Example 3

Patient R., age - 4 years 2 months (study protocol No. 52), applied for admission to the University Clinic of Krasnoyarsk State Medical University of Krasnoyarsk on January 26, 2012. At the initial examination he complained of difficulty in nasal breathing due to poor discharge from the cavity nose, sniffling during night sleep, regardless of body position, frequent acute respiratory viral infections (ARVI) once every 2 months, fatigue. Complaints of a local and general nature within 2 years. Complaints associated with contact with patients with acute respiratory viral infections in a kindergarten. During the year, exacerbation of chronic adenoiditis with prolonged mucopurulent runny nose was verified 6 times. Outpatiently received treatment with a pediatrician and an otorhinolaryngologist with systemic antibacterial drugs, the last time - one month ago. Immunocorrection was not performed. As a result of the treatment, a complete relief of symptoms within 30 days was noted. The reason for applying for a second outpatient appointment was sniffing during a night's sleep amid fatigue.

With endoscopy of the nasal cavity and nasopharynx, cyanosis of the lower nasal concha and hyperplastic pharyngeal tonsil of the II degree are noted. From other ENT organs - signs of pathology were not detected. Lymph nodes of the posterior cervical group are enlarged to 0.7 × 0.7 cm in diameter, of a dense consistency, unsoldered, painless on palpation.

A clinical blood test did not reveal deviations in the number of red blood cells, hemoglobin, with a relative lymphocytosis in the leukocyte formula, a slightly accelerated erythrocyte sedimentation rate (ESR) of 12 mm / hour, which was a laboratory indicator of endogenous intoxication, indirectly reflected the ratio in the blood plasma of protein particles large sizes (globulins) and small sizes (albumin) and testified to the inflammatory process.

The results of a study of the immune status of patient R. showed a significant increase of 8% in the relative number of all mature T-lymphocytes bearing a cluster of CD3 ⁺ cell differentiation in comparison with healthy children, which noted the absence of T-cell immunodeficiency. The values of the remaining indicators did not exceed the established limits of the norm for each of them.

During bacteriological examination of biological material from the surface of the pharyngeal tonsil, the reference value of the degree of contamination was revealed mainly due to the multiculture of opportunistic microflora - Staphylococcus aureus and staphylococcus saprophytic. The quantitative content of microorganisms per 1 g (ml) of the test material amounted to 1 × 10 ³ CFU / g (ml) (CFU - colony forming unit).

According to the radiograph of the paranasal sinuses, no pathology was detected.

Taking into account complaints, anamnesis, objective examination data and a comprehensive clinical, laboratory and instrumental study by an otorhinolaryngologist, allergist and pediatrician, chronic adenoiditis, purulent form, and remission were diagnosed.

A survey on the proposed method. In the peripheral blood lymphocytes of patient R., the relative and absolute values of the functional activity of lymphocyte enzymes were determined: A ₁ (LDH) = 71.41 μE per 10 ⁴ lymphocytes, A ₂ (NADPHDG) = 0.04 μE per 10 ⁴ lymphocytes, A ₃ (MDH) = 0.0001 μE per 10 ⁴ lymphocytes, A ₄ (NADGDG) = 27.44 μE per 10 ⁴ lymphocytes. The value of a significant indicator of the functional activity of lymphocyte enzymes K = 2.6 was calculated and a favorable course of chronic adenoiditis was diagnosed.

At the visit of patient R. 90 days after the initial treatment, relief of general and local symptoms was established. With endoscopy of the nasal cavity and nasopharynx, the mucous membrane of the nasal cavity is pink, pharyngeal tonsil I degree. Lymph nodes of the posterior cervical group 0.5 × 0.5 cm in diameter, elastic consistency, unsoldered, painless on palpation. SARS did not suffer reinfection.

In a clinical blood test, an ESR of 6 mm / h was normalized, indicating a favorable course of the disease and the patient's recovery.

Immunological examination showed that the studied immunological parameters did not exceed the established limits of the norm for each parameter.

In retrospect, the course of chronic adenoiditis has been identified as favorable with the elimination of immuno-metabolic disorders. The coincidence of the determination of the favorable variant of the course of chronic adenoiditis is complete - 100%.

The technical result of the invention consists in a reliable, informative, time-optimized determination of the functional activity of all populations of lymphocytes that form the total immune response, on which an early prospective determination of the course of chronic adenoiditis in children depends.

The technical result consists in the objective diagnosis of indicators due to:

1) the use of the etiological antigenic stimulus of vital lymphocytes as a sole stimulator of proliferative activity of lymphocytes in chronic adenoiditis, taking into account the indicators conventionally called “enzyme” and “substrate background of enzymes”, which excludes an additional background of lymphocyte activity;

2) determining the functional activity of the enzymes of all lymphocytes in the vital state, objectively characterizing quantitative and qualitative changes in the metabolism of lymphocytes, which demonstrates the possibility of using the method for a broad description of the functional activity of lymphocytes in real time, providing high sensitivity;

3) automated determination of the functional activity of lymphocytes with the evaluation of the results, regardless of subjective factors;

4) a high level of coincidence of the prospective and retrospective definition of flow options (94.5%).

The method includes a unified assessment of the quality and quantity of correlation between laboratory parameters with the calculation of a significant indicator of the functional activity of lymphocyte enzymes. The developed test of a significant indicator of the activity of lymphocyte enzymes K <0.9 and K≥0.9 in peripheral blood is informative for the differential diagnosis of metabolic conditions caused by disorders in chronic adenoiditis and other inflammatory diseases. Obtaining a result on the functional activity of lymphocytes with a quantitative characteristic is an important criterion for diagnosis and can be applied at the initial stage of the examination in the absence of changes in the number of cells, as well as at subsequent stages in order to monitor the course.

The technical result from the implementation of the method is to reduce the diagnostic time due to the time of culturing lymphocytes for less than 2 hours, which makes it possible to timely determine the functional state of lymphocytes, adjust the plan and tactics of managing sick children with chronic adenoiditis and improve the results of their rehabilitation.

The technical result of the invention consists in a significant reduction in the cost of laboratory tests due to the low cost of the reagents and equipment used, it is simple to carry out, which ensures widespread use of the proposed method in the initial clinical examination.

The proposed method can be recommended for use in clinical practice by an otorhinolaryngologist, immunologist, pediatrician. The method extends the determination of the functional activity of lymphocytes in chronic adenoiditis in children.

Brief Description of the Drawings

Figure 1 shows

The activity of NAD (F) -dependent dehydrogenases in blood lymphocytes in healthy children and patients with chronic adenoiditis (Me, P ₂₅ -P ₇₅ ):

A comparison of activity indicators of NAD (F) -dependent dehydrogenases in blood lymphocytes in healthy children and patients with chronic adenoiditis, presented as a median with percentile values that the given median does not exceed with a fixed probability;

p - level of statistical significance when comparing indicators with a group of clinically healthy children who formed the control (p <0.05).

Figure 2 shows

The concentration of substrates, cofactors and pH indicators for determining the activity of NAD (F) -dependent dehydrogenases in blood lymphocytes by a bioluminescent method:

concentration of substrates, cofactors, pH values of the buffer medium specific for the enzymes LDH, NADPHDG, MDH and NADGDG, necessary for preparing the incubation mixture when determining the activity of these enzymes, are presented.

Figure 3 shows

The coincidence of the retrospective and prospective determination of the variants of the course of chronic adenoiditis by the value of a significant indicator of the activity of lymphocyte enzymes: the prospective determination of the variant of the course of chronic adenoiditis in 56 children is compared by the value of the significant indicator of the activity of lymphocyte enzymes (K) and the retrospective determination of the course of the course of the relief / absence of relief of disease symptoms after 90 days.

Claims (1)

A method for determining the functional activity of lymphocytes in chronic adenoiditis in children, including the isolation of lymphocytes from venous blood by centrifugation, treatment of the supernatant with a solution of sodium chloride, centrifugation of diluted blood and counting the number of lymphocytes, characterized in that they determine the purity of lymphocyte output by microscopic control of the morphological composition of leukocyte suspensions, then determine the viability of lymphocytes by staining with a solution of trypan blue, conduct OS motic lysis of a suspension of isolated lymphocytes containing cells at a concentration of 1.0 million / ml by introducing distilled water 1: 5 by volume into a suspension of lymphocytes, 1.0-2.0 ml of dithiothreitol, the resulting mixture of destroyed lymphocytes in an amount of 200 μl is incubated with 600 μl of a substrate with a concentration of 0.50-8.70 mm and a cofactor with a concentration of 0.50-8.10 mm and determine the preliminary relative value of the functional activity of the enzymes of lymphocytes by measuring the glow when mixing the incubation mixture and bioluminescent reagents, the absolute value lymphocyte functional activity of enzyme is determined from the calibration curve of fluorescence intensity versus concentration of lymphocytes enzymes and functional activity was determined according to the formula
$$A=\frac{\Delta C\times V\times \mathrm{one}{0}^{-6}}{T},$$

where A is the functional activity of lymphocyte enzymes, E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);
ΔС is the difference in the concentrations of lymphocyte enzymes in absolute and relative values, mmol;
V is the sample volume, in ml;
T is the incubation time, min;
the value of a significant indicator of the activity of lymphocyte enzymes is determined by the formula
$$\mathrm{TO}=\frac{{\mathrm{BUT}}_{\mathrm{one}}+{\mathrm{BUT}}_2}{{\mathrm{BUT}}_3+{\mathrm{BUT}}_{\mathrm{four}}},$$

where K is a significant indicator of the activity of lymphocyte enzymes;
A ₁ - functional activity of the enzyme lactate dehydrogenase (LDH) lymphocytes, E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);
A ₂ - functional activity of the enzyme nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADPHDG), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);
A ₃ - functional activity of the enzyme malate dehydrogenase (MDH), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);
A ₄ - functional activity of the enzyme nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase (NADGDG), E (unit of enzyme activity) per 10 ⁴ lymphocytes (1E = 1 μmol / min);
when the value of a significant indicator of the activity of lymphocyte enzymes (K) is equal to or more than 0.9, the activity of the lymphocytes is evaluated as adequate, which determines the favorable course of chronic adenoiditis, and when the value of a significant indicator of the activity of enzymes of lymphocytes is less than 0.9, the activity of the lymphocytes is determined as reduced, which determines the unfavorable the course of chronic adenoiditis.

Images