RU2242763C1 - Method for detecting phagocytic activity of neutrophils in peripheral blood of alive organisms - Google Patents

Abstract

FIELD: medicine, immunology, allergology, pulmonology, dermatology, infectious microbiology, veterinary science.

SUBSTANCE: the method has been suggested to exclude the possibility for obtaining falsely overestimated and falsely underestimated results of detection due to creating the conditions for keeping functional properties of neutrophils in vitro for every concrete organism. Moreover, one should perform blood sampling with anticoagulant, prepare the mixture to study phagocytosis that contains leukocytes and phagocytosis object in the course of which one should, at first, introduce phagocytosis object into blood with anticoagulant 30 min after its sampling, not later followed by filling in two capillaries with the obtained mixture. Mixture should be treated to study phagocytosis due to incubation at 37 C for every capillary, moreover, one of them should be incubated for 30 min, another one - for 2 h, and due to centrifuging every capillary for 10 min at 1500 rot./min. Right after centrifuging one should isolate leukocytes due to separating each capillary at the border of leukocytic residue and erythrocytic residue sedimentation and removing leukocytic residue out of corresponding part of every separated capillary. Smears should be prepared out of leukocytic residues obtained after centrifuging to determine the number of phagocytosis-entering neutrophils upon every smear and total number of microbes absorbed by neutrophils. Phagocytosis values should be calculated, they are: phagocytic index, phagocytic number, coefficient of phagocytic number and index of neutrophilic bactericidity. The data obtained for phagocytosis parameters should be compared to normal ones to conclude either upon availability or absence of neutrophilic phagocytic activity lesions in peripheral blood. The method increases the number of ways to evaluate phagocytic blood activity.

EFFECT: higher accuracy of detection.

1 cl, 3 ex, 8 tbl

Description

The invention relates to medicine, namely to immunology, allergology, pulmonology, dermatology, infectious microbiology, veterinary medicine, and can be used in assessing the immune status of a healthy and sick body.

A known method for determining the phagocytic activity of leukocytes in patients and healthy people [1]. The formulation of the phagocytosis reaction according to the analogue method is as follows. During the preparation of the mixture for the study of phagocytosis, 0.1 ml of 2% sodium citrate, 0.2 ml of the test blood and 0.1 ml of the suspension of the test microbe are poured into a vidale test tube, using laboratory strain Staphylococcus No. 209 or B. mesenthericus (daily agar culture according to the standard) at a concentration of 400 million microbial bodies in 1 ml (object of phagocytosis). All components are thoroughly mixed, after which the resulting mixture is processed to study phagocytosis. In this case, the tube is placed for 30 min in a thermostat (37 ° C). After incubation, the tube with the mixture is centrifuged for 3 min at 1000 rpm. Then, smears are prepared from the upper layer of the precipitate, which are fixed with a mixture of Nikiforov (equal parts of alcohol and ether) and stained according to Romanovsky-Giemsa. Under the microscope, 100 leukocytes (total leukocyte count) are examined and the total number of microbes absorbed by leukocytes is counted. Calculation of phagocytosis indicators, which use 2 indicators:

1. The percentage of phagocytic leukocytes, i.e. the number of leukocytes out of 100 that showed phagocytic activity (this indicator corresponds in meaning to the indicator "phagocytic index", the definition of which is introduced below in the text when describing the prototype method).

2. Phagocytic number, ie the number of microbes absorbed on average by one white blood cell.

For healthy individuals, the percentage of phagocytic leukocytes is 50-70%, and the phagocytic number is 2.0-4.0.

The known analogue method [1] does not ensure the achievement of the technical result of the claimed method in connection with the following. The time parameter of the incubation mode of the mixture for the study of phagocytosis (30 min), provided by the method of the analogue method, allows only two phagocytosis indicators to be determined: the phagocytic index (after a 30-minute incubation) and the phagocytic number (after a 30-minute incubation). These indicators reflect the absorption capacity of neutrophils at the initial stage of phagocytosis. The inability to study the final stage of phagocytosis (for example, after a 2-hour incubation) makes it impossible to determine such important phagocytosis indicators as the phagocytic number coefficient and neutrophil bactericidal index (to calculate which, in particular, indicators determined on smears prepared from sediments are used 2 hour incubation). The phagocytic coefficient reflects the absorption capacity of neutrophils at the final stage of phagocytosis, and the neutrophil bactericidal index reflects the ability of a phagocyte to digest captured microbes at the final stage of phagocytosis. Both of these indicators are included in the totality of phagocytosis indicators, each of which is necessary, and taken together are sufficient to ensure adequate assessment of the phagocytosis reaction. This is due, in particular, to the following reasons. Violations of the phagocytosis process can be discrete and affect both the complex and isolated stages of phagocytosis (chemotaxis, adhesion to the microbe, absorption, digestion of microbes). In this regard, the lack of taking into account indicators reflecting the process of phagocytosis in dynamics minimizes the probability of detecting violations in the phagocytosis system in a number of cases. In addition, the impossibility of assessing neutrophil bactericidal activity will not allow identifying diseases with predominant bactericidal disorders (such as Chediak-Higashi syndrome and chronic granulomatosis), as well as diseases in which phagocytosis disorders (including bactericidal disorders) can play a leading pathogenetic role (pyoderma; adenoflegmon; abscessed pneumonia; long-term non-healing wounds; postoperative complications; secondary immunodeficiencies caused by drug therapy, etc.). Evaluation of the reaction of phagocytosis in such patients is necessary for the choice of therapy tactics, since the same clinical manifestations may be caused by disorders in other parts of the immune system (such as the compliment system, antibody production). In this regard, the method of the analogue method [1] is fundamentally unsuitable for assessing the phagocytic activity of neutrophils in a number of diseases (deliberately erroneous determination results).

The method of the analogue method [1] does not provide for the preliminary separation of leukocytes and erythrocytes and the isolation of leukocytes as special operations during the preparation of a mixture for the study of phagocytosis. The separation of leukocytes and red blood cells is carried out at the stage of processing the mixture to study phagocytosis by centrifugation of the mixture (i.e., after the interruption of the phagocytosis reaction at the initial stage). The centrifugation mode used in the analogue method (3 min at 1000 rpm) does not provide complete precipitation of leukocytes, a significant part of them remains in the supernatant. So, the experiments of the inventors showed that the dependence of the number of leukocytes in the supernatant on the centrifugation time (at 1000 rpm) is as follows:

According to the similar method, the selection of leukocytes is carried out after centrifugation of the mixture for the study of phagocytosis by aspirating the leukocyte film located on the erythrocyte layer. With a short centrifugation time (3 min), this film is formed primarily by neutrophils that have absorbed microbes (i.e., cells with a higher weight), and neutrophils that have not entered into phagocytosis (with a lower weight) can remain in the supernatant. All this significantly reduces the accuracy of the determination of the phagocytic activity of leukocytes. In addition, the separation of leukocytes from red blood cells by suction does not provide the possibility of isolated isolation of leukocytes - there will always be a significant admixture of red blood cells (experiments of the authors of the proposed method showed that with a similar formulation of the phagocytosis reaction, the admixture of red blood cells in smears can be up to 360 red blood cells per 1 leukocyte). This significantly complicates the microscopic evaluation of the reaction results, thereby increasing its duration. The high likelihood of leukocyte trauma to the bottom and walls of the test tube (when mixing the mixture to study phagocytosis during centrifugation) causes a relatively high percentage of death of these cells, as well as the occurrence of various violations of their functional properties, including properties responsible for phagocytosis, which also reduces the accuracy of the analysis.

Thus, the particularities of the formulation of the phagocytosis reaction using the analogue method [1] determine the limited scope of the methodology (due to the fundamental impossibility of adequately assessing the results of the reaction in a number of diseases), as well as the relatively low accuracy of determining the phagocytic activity of leukocytes in cases where the assessment of phagocytosis with using an analogue method is fundamentally possible.

In modern basic guidelines on immunological research methods [2; 3] it is recommended for a wide laboratory practice to use methods involving the study of the phagocytosis process in full, with an assessment of both the initial and final stages of the reaction. In addition, these guidelines, as a rule, provide for the preliminary separation of red blood cells and white blood cells, followed by the isolation of white blood cells as special operations during the preparation of the mixture for the study of phagocytosis.

Closest to the claimed solution on the basis of essential features is a method for determining the phagocytic activity of neutrophils / FAN / peripheral blood in patients and healthy people [4]. The method adopted for the prototype includes the following stages. Blood is taken with an anticoagulant in the patient being examined. At the same time, 10 ml of blood from a cubital vein is introduced into a sterile test tube with a pre-made solution of 0.6 ml of heparin diluted 1:10. Prepare a mixture for the study of phagocytosis. In this case, first the white blood cells and red blood cells are separated, followed by the release of white blood cells. During this operation, the blood is thoroughly mixed and centrifuged for 10 minutes at 1000 rpm. The plasma, together with a layer of leukocytes, is carefully aspirated and placed in a clean centrifuge tube, adding 5-6 ml of 199 medium. The leukocytes are washed by centrifugation for 10 min at 1000 rpm. The supernatant is removed, and the leukocytes in the sediment are resuspended in medium 199 twice, each time repeating the procedure of "soft" centrifugation. After the last centrifugation, the supernatant and part of the cell suspension are sucked off with a pipette, leaving 0.2 ml of the cell suspension in medium 199 in a centrifuge tube. Then, 0.3 ml of microbial suspension is added to the tube containing 0.2 ml of white blood cells in medium 199, as which use a suspension of staphylococci (daily agar culture of Staphylococcus epidermidis strain 9198 according to the standard) at a concentration of 1 billion microbial cells in 1 ml (phagocytosis object), after which 0.15 ml of a pool of fresh donor serum AB (IV) group (opsonizing factor) is added. The components are mixed by gentle rotation, after which the resulting mixture is processed to study phagocytosis. During this stage, the resulting mixture was thermostated for 30 min at 37 ° C. After this time, add 5 ml of isotonic sodium chloride solution heated to 37 ° C to the test tube, shake and centrifuge for 10 minutes at 1500 rpm. At the end of centrifugation, the supernatant is removed and smears are made from a leukocyte suspension, the remains of which are again placed in a thermostat at 37 ° C to continue incubation for another 90 minutes (total incubation time is 120 minutes). Then the smears are prepared repeatedly from the precipitate of a two-hour (120-minute) incubation.

Smears from leukocyte sediments (after a 30-minute and 120-minute incubation) are prepared on fat-free glass slides. The prepared smears are dried in air, then fixed for 10 min in absolute methyl alcohol and stained with azure-eosin according to Romanovsky-Giemsa. After staining, smears are examined under a microscope in an immersion system: at least 200 cells are counted (total number of neutrophils). The number of neutrophils that have entered into phagocytosis and the total number of microbes absorbed by neutrophils are determined on each smear. Phagocytosis indices are calculated, which are used as follows:

1. Phagocytic index / FI /, determined by the formula:

2. Phagocytic number / PS / - the average number of microbes located in 1 neutrophil (intracellular), which is determined by the formula:

Both indicators (FI and PS) are calculated on smears made after a 30-minute and 120-minute incubation (FI30 and FI120, respectively, FP30 and PS120).

3. The coefficient of phagocytic number (KPF), determined by the formula:

4. The neutrophil bactericidal index / IBN /, determined by the formula:

where IBN-index of bactericidal activity of neutrophils,%,

H _y - the number of microbes killed inside neutrophils;

H _p - the total number of microbes absorbed by neutrophils.

5. Opsonic absorption index / IPR /, determined by the formula:

Compare the obtained values of phagocytosis with normal values, which in accordance with the data of [4] are:

Based on the results of the comparison, the presence or absence of changes in each of the studied phagocytosis indices is established in comparison with the norm. When finding the values of all the studied indicators of phagocytosis within normal limits, they conclude that there are no violations of the FAN in the patient being examined. If there are deviations from the norm of at least one of the studied phagocytosis parameters, a conclusion is drawn about the presence of FAN disorders in this patient.

The prototype method [1] does not allow to obtain a technical result achieved using the inventive method, for the following reasons. The preliminary separation of leukocytes and erythrocytes and the isolation of leukocytes envisaged by the method of the prototype method, on the one hand, provides a relatively high purity of leukocyte isolation, contributing to the creation of optimal conditions for microscopic evaluation of the reaction results. However, on the other hand, preliminary separation of leukocytes and erythrocytes excludes the possibility of forming a erythrocyte cushion during centrifugation, which can serve as a buffer, preventing the leukocytes from contacting the bottom of the tube. In this case, the preliminary separation of blood cells is in itself associated with multiple centrifugation (centrifugation of plasma with leukocytes in the preparation of leukocyte suspensions, repeated centrifugation of leukocyte suspensions during washing). Under these conditions, during each centrifugation of the mixture containing leukocytes (both at the stage of preparation of the mixture for the study of phagocytosis — during the preliminary separation of leukocytes and red blood cells, and at the stage of processing this mixture), in the absence of an “erythrocyte pad” all leukocytes in contact with the walls test tubes settle to its bottom. Injury of leukocytes to the bottom and walls of the tube during centrifugation can lead to the occurrence of violations of their functional properties of varying severity (including properties responsible for phagocytosis), as well as to the death of these cells. In particular, nonphysiological activation of a significant number of white blood cells due to their adhesion to the bottom and to the walls of the tube may occur. The presence in the prototype method of multiple mixing of a mixture containing leukocytes, as a special operation (during preliminary separation of blood cells, before incubating the mixture to study phagocytosis in a thermostat and after incubation), and as a result of involuntary shaking of the tube during manipulation during analysis (when placing the tube in the thermostat and removing it from the thermostat, when placing the tube in a centrifuge and removing it from the centrifuge, when moving the tube from place to place, etc.) is also essential It increases the possibility of traumatic contact of leukocytes with the bottom and the walls of the tube. All this significantly increases the likelihood of obtaining false-raised and false-understated reaction results, significantly reducing the accuracy of determining FAN.

The preliminary separation of leukocytes and red blood cells, during which the autologous blood plasma is removed, leads to the removal of autologous factors contained in the plasma. This necessitates the addition of an opsonizing factor, donor serum, to the phagocytosis test mixture, which, along with factors that improve phagocytosis, may contain cytotoxic factors that damage white blood cells. As a result of this, the functional properties of leukocytes may change (including the suppression or activation of phagocytosis may take place) and their viability may decrease. It can also contribute to the production of pseudo-overestimated and pseudo-underestimated results of the reaction and reduce the accuracy of determining FAN.

The objective of the invention is to provide a method for determining the phagocytic activity of neutrophils of the peripheral blood of living organisms, which eliminates the possibility of obtaining false and false and false results of determination by creating conditions for maintaining in vitro the functional properties of neutrophils of each particular organism responsible for phagocytosis, as well as their viability at a level corresponding to or approaching the level in vivo individually inherent in a given organism, by eliminating the mechanical physicochemical, biological, and / or immunological factors that negatively affect the processes of chemotaxis and adhesion of neutrophils, the processes of absorption and digestion of microbes by them, and also causing the death of these cells.

The problem is solved in that in the method for determining FAN of peripheral blood of living organisms, including blood sampling with an anticoagulant; preparation of a mixture for the study of phagocytosis containing leukocytes and the object of phagocytosis, including the isolation of leukocytes; processing the mixture to study phagocytosis, including incubating it for 2 hours at 37 ° C and centrifuging after 30 minutes of incubation and after 2 hours of incubation for 10 minutes at 1500 rpm; preparation of smears from thirty-minute and two-hour incubation pellets obtained after centrifugation, followed by their fixation and staining; determining on each of the smears the number of neutrophils that have entered into phagocytosis and the total number of microbes absorbed by neutrophils, the calculation of phagocytosis indices and the comparison of the obtained values of phagocytosis indices with normal values, according to which it is concluded that there are violations of the FAN of the studied peripheral blood, according to the invention during the preparation of a mixture for the study of phagocytosis, the object of phagocytosis is first introduced into the blood with an anticoagulant no later than 30 minutes after its collection, after whereupon two capillaries are filled with the obtained mixture for the study of phagocytosis. Processing the mixture for the study of phagocytosis is carried out by incubating at a temperature of 37 ° C each of the capillaries containing the specified mixture, one of the capillaries being incubated for 30 minutes and the other for 2 hours, and centrifuging each of these capillaries for 10 minutes at 1500 rpm The selection of leukocytes is carried out immediately after centrifugation by separating each of the capillaries at the boundary of the placement of the leukocyte sediment and the erythrocyte sediment and extracting the leukocyte sediment from the corresponding part of each of the disconnected capillaries. The preparation of smears is carried out from the precipitation of leukocytes obtained from each of the disconnected capillaries. As indicators of phagocytosis, a phagocytic index, a phagocytic number, a phagocytic number coefficient and a neutrophil bactericidal index are used. The most effective is the use during preparation of a mixture for the study of phagocytosis and its processing of capillaries with an inner diameter of 0.2-0.8 mm.

The selection and justification of the optimal characteristics of the causally significant parameters of the claimed method (i.e., parameters that affect the achievement of the technical result) was carried out as follows. As causally significant parameters were considered:

the time from the moment of blood sampling from a living organism to the introduction of the object of phagocytosis into the blood with an anticoagulant (a quantitative parameter that reflects the condition for the action);

modes and conditions for processing the mixture for the study of phagocytosis and subsequent isolation of leukocytes as the main factor providing the possibility of separation of leukocytes and erythrocytes directly during the phagocytosis reaction, as well as as a factor in optimizing the complex effect with respect to the conditions for maintaining the functional properties of neutrophils, including responsible for phagocytosis, and the viability of these cells.

интервала, установленная по результатам хронометрирования авторов изобретения, представляла собой минимальное время, необходимое для доставки крови обследуемого ребенка из процедурного кабинета в лабораторию и последующего отбора образцов крови с антикоагулянтом из исходной пробирки в опытные. Таким образом, исследование фагоцитоза производили с использованием образцов крови с различными сроками хранения (перед внесением объекта фагоцитоза). Заполняли полученной в каждой из опытных пробирок смесью для исследования фагоцитоза по два капилляра с внутренним диаметром 0,8 мм и дальнейшую обработку указанной смеси, приготовление мазков и определение клеточных показателей, необходимых для последующего расчета показателей фагоцитоза, проводили в соответствии с методикой заявленного способа.The choice of the optimal time from the moment of blood sampling from a living organism to the introduction of an object of phagocytosis into the blood with an anticoagulant was carried out by varying the time parameter against the background of optimal characteristics of other parameters of the claimed combination (i.e., under optimal conditions and conditions for performing actions at the stage of processing the mixture to study phagocytosis and white blood cell isolation). At the same time, from 20 practically healthy children aged 3 to 15 years, 0.6 ml of blood was taken from a finger and introduced into test tubes (initial) with a pre-added 5% solution of sodium citrate (in the amount of 0.15 ml per tube) . From each initial tube (containing the blood of one child), 6 blood samples with an anticoagulant (0.1 ml) were taken into separate tubes (6 test tubes). In each of the test tubes containing a blood sample with an anticoagulant, 0.05 ml of a suspension of staphylococci (daily agar culture of Staphylococcus epidermidis pcs. 9198 by standard) was added at a concentration of 1 billion microbial cells per 1 ml (phagocytosis object) at various times from the moment blood sampling from the examined child: in the 1st, 2nd, 3rd, 4th, 5th and 6th test tubes, respectively, after 5, 15, 30, 45, 60 and 120 min after blood collection . The lower boundary (5 min) of the investigated

the interval established by the timekeeping of the inventors was the minimum time required for delivery of blood of the examined child from the treatment room to the laboratory and subsequent sampling of blood with an anticoagulant from the original test tube. Thus, the study of phagocytosis was performed using blood samples with different storage periods (before introducing the object of phagocytosis). Two capillaries with an inner diameter of 0.8 mm were obtained with a mixture for studying phagocytosis in each of the test tubes and further processing of this mixture, preparation of smears and determination of cellular parameters necessary for the subsequent calculation of phagocytosis parameters were carried out in accordance with the method of the claimed method.

For each of the test tubes, FIs (FI30 and FI120) were determined by formula (1) and PS (PS30 and PS120) according to formula (2) (resulting indicators for comparative analysis), compared them with normal values given in [4], and establish the presence or absence of changes in each of the studied indicators of phagocytosis in comparison with the norm.

In addition, neutrophil viability was evaluated by the trypan blue exclusion method as described in [5, p. 50]. In this case, 10 μl of the leukocyte suspension obtained after the separation of each of the capillaries of the 30- and 120-minute incubation periods (for each blood sample) was mixed with 10 μl of a 0.2% trypan blue solution on a glass slide, covered with a coverslip and placed under the microscope. The number of blue cells (dead cells) and unpainted cells (living cells) was calculated as a percentage of the total number of cells counted (at least 100 cells were counted). For each of the test tubes (blood samples), the average value of the indicated indices obtained for the capillaries of 30- and 120-minute incubation periods was calculated. The percentage indicator (average value) of dead neutrophils from the total number of counted cells was used as the resulting indicator for comparative analysis.

Statistical processing of the results was carried out using the Wilcoxon-Mann-Whitney test [6].

The research results are shown in table 1.

From the data of Table 1 it can be seen that in blood samples in which the time between the blood sampling of the examined child and the introduction of the phagocytosis object into the blood with an anticoagulant varied from 5 to 30 minutes, the values of the studied phagocytosis indices (FI30, FI120; PS30, PS120) were within the range of normal values with a minimum percentage of dead neutrophils (1.1-2.3%). At the same time, there were no statistically significant differences between the values of the considered parameters (phagocytosis indicators, percentage of dead neutrophils) of blood samples with storage periods of 5, 15 and 30 minutes (statistical processing of data using the Wilcoxon-Mann-Whitney test). When using blood samples with an anticoagulant stored for more than 30 minutes during the preparation of a mixture for the study of phagocytosis, there was a statistically significant decrease compared with the norm of all analyzed phagocytosis parameters, as well as a statistically significant increase in the percentage of dead neutrophils (6.1-7.5%) . It was found that the differences between the values of each of the considered parameters, determined during the reaction with blood samples of the previous and subsequent storage periods, become statistically significant, starting with blood samples of 30-45-minute storage periods.

The selection and justification of the modes and conditions for performing actions at the stage of processing the mixture for the study of phagocytosis, as well as the action of “leukocyte isolation” was carried out as follows.

Comparative studies of the FAN, determined under various modes and conditions of the formulation of the phagocytosis reaction, were conducted in the following children:

- in children with confirmed by clinical and laboratory examination violations of phagocytosis aged 3 to 12 years (8 people);

- in practically healthy children aged 3 to 15 years (20 people).

The distribution of the examined children into groups was as follows:

Group 1 - patients with Chediak-Higashi syndrome (4 people);

Group 2 - patients with chronic granulomatosis (2 people);

3 group - patients with Job syndrome (2 people);

4 group - practically healthy children (20 people).

According to the data of [7], Chediak-Higashi syndrome refers to congenital immunodeficiencies accompanied by defects in phagocytosis, which are characterized by a defect in neutrophil granules, a significant decrease in neutrophil chemotaxis, and a significant decrease in neutrophil bactericidal activity.

Patients with Chediak-Higashi syndrome (who made up the 1st group) were identified during examination in a hospital of the Children's City Hospital No. 1 of St. Petersburg (hereinafter referred to as Children's Hospital No. 1) for recurrent serious infections (all patients had recurrent pneumonia, otitis media) . The diagnosis was confirmed by a characteristic clinical symptom complex: albinism, nystagmus, neutropenia, giant granules in white blood cells, recurrent severe infections. In the study of chemotaxis of peripheral blood leukocytes according to the method described in [2, p.333-338], a sharp decrease was found in all patients compared with the norm. When a blood test was performed using a test with nitro-blue tetrazolium, as described in [3, p. 87-88], all patients showed a sharp decrease (compared with the norm) in the recovery of nitro-blue tetrazolium, which indicated a profound defect in neutrophil bactericidal activity.

According to the data of [7], chronic granulomatosis refers to congenital X-linked immunodeficiencies, which are characterized by a decrease in the bactericidal activity of neutrophils and a decrease in their release of active oxygen radicals.

Patients with chronic granulomatosis (2 boys who made up the 2nd group) were identified during examination in hospital No. 1 for recurrent abscesses of soft tissues and internal organs (one patient died after a fourth recurrence of liver abscesses 4 years after diagnosis). In both patients, a blood test using a nitro-blue tetrazolium test [3] showed a sharp decrease in neutrophil bactericidal activity compared to normal. When studying chemotaxis according to the technique [2], no abnormalities were found in any of the patients. In addition, the diagnosis of chronic granulomatosis in a deceased patient was confirmed by autopsy.

Job's syndrome, according to [7], refers to congenital immunodeficiencies accompanied by defects in phagocytosis. The vast majority of patients are characterized by a decrease in neutrophil chemotaxis with normal neutrophil bactericidal activity and the release of active oxygen radicals by them.

Patients with Job syndrome (who made up the 3rd group) were identified during a hospital examination of Children's hospital No 1 regarding “cold” abscesses of the subcutaneous tissue, recurrent lymphadenitis, adenoflegmon, not accompanied by skin hyperemia and an increase in skin temperature; recurrent synopulmonary infections; atypical (staphylococcal) eczema. When studying the chemotaxis of peripheral blood leukocytes according to the method [2], a sharp decrease was revealed in both patients. Deviations of the test with nitro-blue tetrazolium [3] in comparison with the norm were not found in any of the patients.

The fourth group consisted of 20 children who did not have any abnormalities in their state of health, either during medical history or during examination (during preventive examinations on the basis of outpatient department No. 41 of Children's City Hospital No. 1) by various clinical, instrumental, and laboratory methods. This group is called "practically healthy children." Indicators of chemotaxis and test with nitro blue tetrazolium, determined by the methods of work [2; 3], all children of the 4th group were within the range of normal values.

During the determination of FAN, 10 ml of blood from a vein was taken from the examined children and introduced into test tubes (initial) with a pre-made 5% sodium citrate solution (in the amount of 2.5 ml per tube). The obtained blood with an anticoagulant was used for staging three series of experiments:

I series of experiments - actions at the stage of preparation of the mixture for the study of phagocytosis, at the stage of processing the specified mixture (incubation, centrifugation), as well as the action of "isolation of leukocytes" was performed using the modes and conditions provided for by the method of the claimed method; capillaries with an inner diameter of 0.8 mm were used as working containers during the preparation and processing of the mixture for the study of phagocytosis followed by the isolation of leukocytes;

II series of experiments - actions at the stage of preparation of the mixture for the study of phagocytosis and at the stage of processing the specified mixture (incubation, centrifugation) were performed using the modes provided by the method of the claimed method; the action of “isolation of leukocytes” was performed after centrifugation of the mixture for the study of phagocytosis (according to the method of the claimed method) using the techniques provided by known methods (prototype method [4], analogue method [1], etc.); as working tanks during the preparation and processing of the mixture for the study of phagocytosis followed by the isolation of leukocytes, 10 ml centrifuge tubes were used;

III series of experiments - actions at the stage of preparation of the mixture for the study of phagocytosis (including the separation of leukocytes and erythrocytes and the isolation of leukocytes) and at the stage of processing the mixture (incubation, centrifugation) were performed using the modes and conditions provided for by the method of the prototype method; 10 ml centrifuge tubes were used as working containers during the preparation and processing of the mixture for the study of phagocytosis.

For statement I of a series of experiments, 0.1 ml was taken from each initial tube (containing the blood of one examined child) into a separate (experimental) tube. 0.05 ml of staphylococcus suspension (daily agar culture of Staphylococcus epidermidis pcs. 9198 by standard) at a concentration of 1 billion microbial cells in 1 ml (object phagocytosis). Two capillaries with an inner diameter of 0.8 mm (working containers) were filled with the obtained mixture for phagocytosis research and further processing of this mixture, preparation of smears and determination of cellular parameters necessary for the subsequent calculation of phagocytosis indices was carried out in accordance with the method of the claimed method. According to the results of the timing of the inventors, the time taken to count on the smears the cellular indices necessary for the subsequent calculation of phagocytosis indices / hereinafter referred to as “the time for counting the cellular indices” / (resulting indicator for comparative analysis) was additionally determined.

For each of the test tubes, FIs (FI30 and FI120) were determined by the formula (1), PS (PS30 and PS120) according to the formula (2), PSP according to the formula (3) and IHB according to the formula (4) (resulting indicators for comparative analysis) . The obtained values of the studied phagocytosis parameters were compared with the normal values given in [4], and according to the results of the comparison, they judged (as described in the methodology of the claimed method) the presence or absence of peripheral blood FAN disorders (phagocytosis disorders) in each of the examined children. The number of children with identified phagocytosis disorders was used as the resulting indicator for the comparative analysis.

When viewing smears under a microscope in an immersion system, along with the cellular indices necessary for the subsequent calculation of phagocytosis indices, the total number of red blood cells in the viewed fields of view was additionally calculated and the number of red blood cells per 1 neutrophil was determined (the resulting indicator for comparative analysis).

In addition, for each of the test tubes, the viability of neutrophils was estimated by the trypan blue exclusion method according to the method of [5], similar to that described above during the experiment for choosing the optimal values of a causally significant time parameter. The percentage of dead neutrophils from the total number of counted cells was used as the resulting indicator for comparative analysis.

For statement of the II series of experiments from each source tube (containing the blood of one child) were selected 0.1 ml in a separate (experimental) tube. 0.05 ml of staphylococcus suspension (the culture is the same as in the first series of experiments) at a concentration of 1 billion microbial cells in 1 ml (phagocytosis object) was added to each of the test tubes containing blood with an anticoagulant 10 minutes after blood sampling from the subject. ) and mixed the components. The resulting mixture for phagocytosis assay was distributed into 2 10 ml centrifuge tubes (working tubes) and each of the working tubes was incubated at 37 ° C: the first for 30 minutes, the second for 120 minutes (2 hours). At the end of the incubation period, the contents of each of the working tubes were mixed, then each of the working tubes was centrifuged for 10 min at 1500 rpm. After centrifugation, a film of leukocytes from the surface of the erythrocyte sediment was aspirated with a Pasteur pipette, transferred to a defatted glass slide and a smear was prepared. Preparation of smears and determination of cellular parameters necessary for the subsequent calculation of phagocytosis indicators was carried out in accordance with the method of the claimed method.

The resulting indicators for a comparative analysis: indicators of phagocytosis (FI, PS, CPF, IBN); the number of children with identified violations of phagocytosis; the percentage of dead neutrophils from the total number of counted cells; the number of red blood cells per 1 neutrophil; the time for counting cellular indicators was determined similarly to that described above for series I experiments.

To set up the III series of experiments, the remaining blood in each of the initial tubes (containing the blood of one child) was mixed and centrifuged for 10 min at 1000 rpm. Plasma with a layer of leukocytes was aspirated, pipetted into a clean centrifuge tube, and 10 ml of 199 medium was added. The leukocytes were washed twice in medium 199 by centrifugation twice for 10 min at 1000 rpm. After the last centrifugation, the supernatant was aspirated, the leukocyte pellet was resuspended in 199 medium to a concentration of 10 × 10 ⁶ cells / ml, and 0.2 ml of the obtained leukocyte suspension was introduced into two 10 ml centrifuge tubes (working tubes). 0.3 ml of suspension of staphylococci (the culture is the same as the first series of experiments) at a concentration of 1 billion microbial cells in 1 ml (phagocytosis object) was added to each of the working tubes, after which 0.15 ml of a pool of fresh AB donor serums was added ( IV) groups (opsonizing factor) (the time from the moment of blood sampling from the examined child to the introduction of the object of phagocytosis into the leukocyte suspension amounted to 35-45 min according to the results of timekeeping of the inventors). The components were mixed and each of the test tubes was incubated at a temperature of 37 ° C: the first for 30 minutes, the second for 120 minutes (2 hours). At the end of the incubation period, 5 ml of isotonic sodium chloride solution heated to 37 ° С was added to each of the working tubes, shaken and centrifuged for 10 min at 1500 rpm. Simultaneous incubation and centrifugation of working tubes (instead of the prototype method provided for in the sequential implementation of the indicated actions) was introduced to ensure the adequacy of stable (i.e. not subject to variation) conditions for the formulation of the phagocytosis reaction in all three series of experiments. The specified modification does not adversely affect the values of the analyzed resulting indicators.

Preparation of smears and determination of cellular parameters necessary for the subsequent calculation of phagocytosis indicators was carried out in accordance with the method of the claimed method.

The resulting indicators for a comparative analysis: indicators of phagocytosis (FI, PS, CPF, IBN); the number of children with identified violations of phagocytosis; the percentage of dead neutrophils from the total number of counted cells; the number of red blood cells per 1 neutrophil; the time for counting cellular indicators was determined similarly to that described above for series I experiments.

Statistical processing of the results in all three series of experiments was carried out using the Wilcoxon-Mann-Whitney, Fisher criteria [6].

The research results are presented in table.2, 3.

From the data of table 2 it can be seen that the use of the claimed method (series I experiments) made it possible to identify phagocytosis defects in all patients in whom these defects were previously confirmed during clinical and laboratory examination. Moreover, using the proposed method, defects of those phagocytosis links were revealed which, as follows from the literature [7], are characteristic of the above diseases. So, in all patients with Chediak-Higashi syndrome (group 1), a significant decrease was found in comparison with the norm of indicators of FI (FI30 and FI120), PS (PS30 and PS120) and IHD (differences are statistically significant) with a slight (compared normal) a decrease in KPh (differences are not statistically significant). In chronic granulomatosis (group 2) in both patients was observed compared with normal values: a significant decrease in IHD (differences are statistically significant); a slight decrease in FI30 (differences are not statistically significant) and a more significant decrease in FI120 (differences are statistically significant); decrease in KFCh (differences are statistically significant). In the 3rd group in all patients with Job syndrome, the results of determination using the inventive method showed a significant decrease in comparison with the norm of FI30 and FI120 (the differences are statistically significant) with a slight decrease in PS30, PS120, KPH and IBN (the differences are not statistically significant). At the same time, the results of the examination of practically healthy children using the claimed method showed the absence of violations of the FAN: in all children of the 4th group, all the analyzed phagocytosis indices were within the range of normal values.

In the II series of experiments, false-elevated indicators of phagocytosis were obtained:

- in three patients of the 1st group and in one patient of the 3rd group - FI30, FI120, FC30 and FC120 (the differences compared with normal values are statistically significant);

- in two patients of the 1st group and in one patient of the 2nd group - IHD (differences are statistically significant);

- in one patient of the 2nd group and in one patient of the 3rd group - KFCH (differences are statistically significant).

This did not allow revealing defects of phagocytosis in three of four patients of the 1st group, in one of two patients of the 2nd group and in one of two patients of the 3rd group.

In the 4th group (practically healthy children), four children received false-enhanced indicators: FI30, FI120, ФЧ30, ФЧ120, КФЧ, ИБН and in one child - false-lower indicators: ФИ30, ФЧ30 and IBN (in all cases, differences compared to normal values are statistically significant). Moreover, none of these children has any diseases and / or various kinds of diagnostic, therapeutic and / or preventive procedures that could cause an increase (intercurrent infections, purulent-inflammatory diseases, injuries, operations, chronic foci of infection, prophylactic vaccinations and etc.) either a decrease (congenital or acquired defects in phagocytosis; X-ray irradiation; treatment with hormones, cytostatics, etc.) of phagocytosis indices in history (within 6 months before determining FAN), nor When observing the dynamics of children (within 2 months after the determination of FAN) were observed.

In the III series of experiments, false-elevated phagocytosis indices were obtained:

- in two patients of the 1st group and in one patient of the 3rd group - FI30, FI120, PCh30 (the differences compared with normal values are statistically significant);

- in one patient of the 1st group - FC120 (differences are statistically significant);

- in two patients of the 1st group, in one patient of the 2nd group and in one patient of the 3rd group - KFCH (differences are statistically significant);

- in two patients of the 1st group and in one patient of the 3rd group - IHD (differences are statistically significant).

This did not reveal violations in the phagocytosis system in two of the four patients of the 1st group, in one of the two patients of the 2nd group and in one of the two patients of the 3rd group.

In the 4th group, five children received false-overestimated indicators: FI30, FI120, ФЧ30, ФЧ120, КФЧ, ИБН and two children - false-lower indicators: ФИ30, ФЧ30, and IBN (in all cases, the differences compared to normal values are statistically significant). Moreover, none of these children, like in the second series of experiments, had any diseases and / or diagnostic, therapeutic and / or preventive procedures that could cause disturbances in the phagocytosis system, neither in history, nor during observation in dynamics (with observation periods similar to the second series of experiments). The data in Table 3 show that the formulation of the phagocytosis reaction using the stated modes and conditions (series I experiments) provides a minimum percentage of neutrophil death (2.3-2.5%) compared with series II and III experiments. At the same time, the purity of the leukocyte isolation, which is judged by the quantitative value of the erythrocyte impurity (defined as the number of red blood cells per 1 neutrophil), and, accordingly, the time spent on the smear counting of the cellular indices necessary for the subsequent calculation of phagocytosis indices, in series I experiments are comparable with similar indicators of the III series of experiments, i.e. under the conditions and conditions for setting the phagocytosis reaction provided for by the method of the prototype method (differences in the values of these indicators are statistically unreliable). The modes and conditions for the formulation of the phagocytosis reaction in the II series of experiments provide a significantly lower degree of purity of leukocyte isolation - the admixture of erythrocytes exceeds the corresponding indicator of the I series of experiments by 288-302 times. This leads to an increase in 3.6-4.2 times the time spent on the calculation of smears of cellular indicators necessary for the subsequent calculation of phagocytosis (compared with the same time indicator of a series of experiments).

Experimental studies of the inventors have shown that the use of phagocytosis of capillaries with an inner diameter of 0.2-0.8 mm during the formulation of the reaction is optimal, because provides the greatest simplicity of the technical implementation of the operation "allocation of leukocytes" in the absence of cases of obtaining false or false lower reaction results. When using capillaries with an inner diameter exceeding 0.8 mm, there were technical difficulties during the operation "leukocyte isolation". In this case, when the capillary was separated, spontaneous (and not induced by touching the tip of the capillary to the glass slide) leakage of its contents into the external medium occurred. This led to an increase in plasma impurity to the leukocyte sediment when transferring it to a glass slide, which reduced the density of the leukocyte suspension and thereby increased the duration of the microscopic evaluation of the reaction results by 2–3 times. In addition, when using capillaries with an inner diameter of 1.0 and 1.2 mm, false-increased rates of phagocytosis were obtained in 6-10% of cases and false-lowering rates in 5-7% of cases. The use of capillaries with an inner diameter of less than 0.2 mm (0.1 mm) led in some cases (especially in patients with leukopenia, granulocytopenia, etc.) to obtain smears of poor quality - in 10-15% of cases with a microscopic analysis of all less than 200 neutrophils were detected by smear. When setting up the phagocytosis reaction using capillaries with an internal diameter of 0.1 mm, false-lower phagocytosis rates were obtained in 4-7% of cases, and false-raised ones in 4-5% of cases.

The achievement of the technical result provided by the invention is due to the following. It is known that in order to obtain the most accurate results of immunological reactions (including the phagocytosis reaction), it is necessary to create conditions for maximally maintaining the in vitro state of the cells corresponding to their in vivo state. In particular, it is known that cell damage during centrifugation occurs upon contact with the bottom of the tube, whereas cells in a suspension are not damaged even under severe centrifugation conditions (several thousand g) [5]. Optimization of the conditions for the formulation of these reactions can be achieved using various technical means and techniques (used both in isolation and in combination), in particular, due to:

- the use of working containers during the reaction formulation, which allows minimizing (or even completely eliminating) the contact of cells with rough surfaces (tubes made of low-adhesive plastic, for example polypropylene, etc.);

- careful handling of cell suspension, which reduces mechanical damage to cells;

- the presence in the incubation medium of factors (autologous or heterologous) that contribute to the preservation of cell viability and their functional properties, etc. [3].

However, when analyzing the patent and scientific and medical literature, the inventors did not find methods for setting up the phagocytosis reaction, where the used technical means and techniques would provide the possibility of dividing the blood of the examined organism into shaped elements directly during the phagocytosis reaction, and the role of this modification would be revealed and proved methods as a factor in optimizing the complex effect with respect to conditions ensuring the preservation of the in vitro functional properties of neutrophils are responsible x for phagocytosis, at a level corresponding to the level in vivo individually inherent in a given organism.

The procedure for determining FAN in such a way that the separation of leukocytes and erythrocytes occurs directly during the phagocytosis reaction was made possible by processing the mixture to study phagocytosis (incubation, centrifugation stages) not in a test tube, but in a capillary. A feature of capillaries is the formation (when they are filled with liquid) on the inner side surface (walls) of the capillary of a stable layer of liquid, which is retained on the walls due to surface tension forces (wall layer of the capillary). In the parietal layer of the capillary, according to the inventors, the blood cells are separated according to the technology characteristic of liquid chromatography, described, for example, in [8]. In this case, the glass wall of the capillary functions as a glass chromatographic carrier used in practice, in particular, for separating a mixture of thermally unstable substances by liquid chromatography [8]. Under the conditions of the claimed method, the formed blood elements under the influence of gravitational forces "slide" towards the bottom of the capillary along the parietal layer, which, acting as a "sliding surface", simultaneously provides braking of these elements. Given the different adhesive ability of blood cells, the degree of inhibition of these elements and, accordingly, their speed of movement are significantly different. In addition, given that the parietal layer of the capillary forms a closed circular surface, the motion vector of the formed elements, in particular leukocytes, is directed down (to the bottom of the capillary), while traumatic contact of cells with the walls of the capillary is completely eliminated. All this creates optimal conditions for the separation of leukocytes and erythrocytes and determines that the process of separation of leukocytes and red blood cells already at the stage of incubation of the mixture for the study of phagocytosis proceeds with relatively high intensity. In this case, chromatographic effects (occurring during the reaction in the capillary), a significantly smaller capillary bottom area compared to the bottom of the tube (with equal volumes of the mixture contained in the indicated containers for phagocytosis studies), the absence of mixing of the capillary contents (as a special operation and as a result of involuntary shaking during manipulation during the analysis), already at the incubation stage, they ensure synchronization of the processes of separation of blood cells and the formation of erythrocyte second cushion "and their continuity. In this regard, the erythrocyte pillow formed at the incubation stage has high stability, density and height (experiments of the inventors on the justification and conditions for the formulation of the phagocytosis reaction: series I experiments).

When setting the reaction of phagocytosis in a test tube, subject to other conditions and modes provided for by the method of the claimed method (experiments of the inventors to substantiate the modes and conditions for setting the reaction of phagocytosis: series of experiments II), at the incubation stage, the process of separation of blood into shaped elements is very slightly expressed (especially in initial incubation period). At this stage, there are some signs of the beginning of the process of formation of the erythrocyte pillow. However, due to the absence of the above factors specific for the claimed method (lack of chromatographic effects, relatively large bottom area of the tube, the presence of mixing — as a special operation — before incubation and after incubation, as well as as a result of involuntary shaking of the tube during manipulation during analysis), the formation process erythrocyte pillow is discrete in nature, there is no synchronization of the processes of separation of white blood cells and red blood cells and the process of formation of "eri a three-piece pillow. " As a result, the erythrocyte cushion formed during the incubation stage is extremely unstable, has a low density, and its height (by the end of the incubation period) is 20-30 times lower than that achieved when the phagocytosis reaction in the capillary was formulated.

When the reaction of phagocytosis in the capillary is set at the centrifugation stage, where there is a sharp intensification of the process of separation of blood cells, the erythrocyte pillow is even more compacted, while there is no possibility of its displacement relative to the bottom of the capillary. Under these conditions, leukocytes during centrifugation settle on a stable, dense and high “erythrocyte cushion”, which completely excludes the possibility of contact of these cells with the capillary bottom, as well (taking into account parietal effects) and with the walls of the capillary. This, in turn, minimizes the likelihood of leukocyte trauma to the bottom and walls of the capillary, thereby preventing the death of neutrophils, as well as the occurrence of various kinds of violations of their functional properties, including properties responsible for phagocytosis (i.e., properties that determine the intensity of chemotaxis and adhesion of these cells, the processes of absorption and digestion of microbes by them). In particular, the absence of contact with the bottom and with the walls of the capillary excludes the possibility of nonphysiological activation of neutrophils, including non-physiological activation of their phagocytic function due to adhesion to the bottom and wall of the capillary.

When a phagocytosis reaction is set up in a test tube using the modes and conditions provided for by the second series of experiments, the process of separation of leukocytes and red blood cells proceeds mainly at the centrifugation stage. At the same stage, the formation of the erythrocyte pillow becomes more pronounced (than at the incubation stage). However, taking into account the later (than in the capillary) periods of formation of this substance and the technical features of the tubes (bottom area, volume), the erythrocyte pillow formed in the tube as a result of centrifugation has a significantly lower density, lower height, and significantly lower stability (compared with the erythrocyte pillow of the capillary), which leads to a relatively high probability of its displacement relative to the bottom of the tube. Under these conditions, the possibility of leukocyte contact with the bottom and with the walls of the tube increases significantly, which, in turn, increases the likelihood of leukocyte trauma, leading to an increase in the percentage of death of neutrophils, to impaired functional properties, in particular, to changes in their phagocytic activity ( suppression or non-physiological activation of phagocytosis).

Thus, the formulation of the phagocytosis reaction in the capillary using the modes and conditions of the claimed method, allowing you to eliminate a number of mechanical and physico-chemical factors that adversely affect the FAN and their viability, significantly reduces the likelihood of obtaining false and false lower reaction results, thereby increasing the accuracy of determination .

Centrifugation of a mixture for the study of phagocytosis in a capillary (I series of experiments) also provides a higher quality of separation of blood cells compared to centrifugation in vitro (II series of experiments). The centrifugation of the leukocyte sediment in the capillary in the form of a column, the height of which exceeds the similar indicator obtained when using the test tube, is 40-50 times, the presence of a clearly defined interface between leukocytes and red blood cells, as well as a more stable erythrocyte pillow, allows separation leukocyte sediment from erythrocyte sediment by a simple fracture of the capillary. This greatly simplifies the technical implementation of the operation "allocation of white blood cells." In this case, the purity of the isolation of leukocytes (which is judged by the quantitative value of the erythrocyte impurity in smears) in the first series of experiments is comparable to the same indicator obtained when the phagocytosis reaction was performed using previously isolated (by the method of the prototype method) leukocytes (III series of experiments). This, in turn, ensures the comparability of the conditions and duration of the microscopic evaluation of the results of the phagocytosis reaction when it is set according to the method of the claimed method and the prototype method (I and III series of experiments).

The leukocyte sediment obtained during centrifugation of a mixture for studying phagocytosis in vitro (series II experiments) is an unstable thin layer on the surface of an erythrocyte sediment, the boundary between leukocytes and erythrocytes is much less pronounced, and the erythrocyte pillow is less stable than in a capillary. Under these conditions, the separation of leukocytes from red blood cells by a simple rupture of a tube (similar to a rupture of a capillary) is technically impossible. The separation of the leukocyte sediment from the erythrocyte sediment by suctioning the leukocyte layer does not provide the possibility of isolating leukocytes with a degree of purity necessary and sufficient to create optimal viewing conditions for smears. A significant admixture of red blood cells in smears significantly complicates the microscopic assessment of the results of the phagocytosis reaction and increases its duration by 3.6-4.2 times (compared with the first series of experiments).

Thus, the formulation of the phagocytosis reaction in capillaries allows the process of separation of leukocytes and red blood cells directly during this reaction (at the stage of incubation of the mixture to study phagocytosis), followed by its completion at the centrifugation stage, and the operation “isolation of leukocytes” after the completion of the phagocytosis reaction (after centrifugation). Moreover, for the process of separation of white blood cells and red blood cells does not require special additional operations (as opposed to the prototype method); the operation “isolation of leukocytes” is relatively simple in technical implementation, and the conditions and duration of the microscopic evaluation of the results of the phagocytosis reaction during the implementation of the claimed sequence of actions (in combination with the modes and conditions of their execution) are comparable with similar indicators obtained during the implementation of the sequence of actions (in combination with modes and conditions for their implementation) of the prototype method. Thus, the claimed sequence of actions provides the relative simplicity of the technical execution of the procedure for determining the FAN and allows to reduce its total duration by 30-40 minutes compared with the prototype.

The use of the whole blood of the examined organism as one of the components of the mixture for studying phagocytosis of the examined organism, separation of leukocytes and erythrocytes directly during the phagocytosis reaction, and isolation of leukocytes after the completion of the phagocytosis reaction stipulate the presence of blood plasma and red blood cells in the reaction mixture as one of the components of the claimed method. On the one hand, this allows you to save autologous factors contained in the blood plasma, contributing, in the opinion of the inventors, to the preservation of the functional properties of neutrophils, including their phagocytic activity, as well as their viability. Under these conditions, the need for introducing an opsonizing factor, a donor serum, which, according to the authors of the claimed method, may contain cytotoxic factors that damage neutrophils, is excluded from the mixture for the study of phagocytosis. This eliminates the possibility of violations of the phagocytic function of neutrophils, as well as their viability, due to the negative effects of these immunological factors.

On the other hand, the presence of red blood cells in a mixture for the study of phagocytosis from the moment the phagocytosis reaction begins, according to the authors of the proposed solution, can also help maintain the functional properties of leukocytes and their viability in view of the following. It is known that red blood cells have a large sorption surface, as a result of which various substances can be adsorbed on their surface [9], including biologically active substances, such as chymokines and cytokines, as well as microbes (in particular, staphylococci). The inventors suggest that biologically active substances adsorbed on the surface of an erythrocyte (such as granulocyte-macrophage colony stimulating factor; interleukins: IL-1, IL-2, IL-3, IL-6, IL-10, IL-18, etc. p.) can positively affect the functional properties of white blood cells (including those responsible for phagocytosis) and their viability both with direct contact of white blood cells and red blood cells, and as a result of the release of these biologically active substances in the reaction medium. In addition, according to the inventors, staphylococci adsorbed on the surface of red blood cells are more likely to undergo a phagocytosis process than staphylococci in solution.

Thus, the sequence of actions of the claimed method in combination with the qualitative composition of the mixture for the study of phagocytosis eliminates immunological factors that can negatively affect the phagocytic function of neutrophils and their viability, thereby contributing to the preservation of the functional properties of neutrophils, including responsible for phagocytosis. It also contributes to reducing the likelihood of obtaining false and false lower reaction results and, accordingly, improves the accuracy of determining FAN.

параметра, позволяет исключить возможность нарушений фагоцитарной функции нейтрофилов, а также снижения их жизнеспособности, обусловленных негативным воздействием указанного биологического фактора, что значимо снижает вероятность получения ложнозавышенных и ложнозаниженных результатов реакции.Preparation of a mixture for the study of phagocytosis, taking into account the optimal shelf life of the blood of the examined organism (with an anticoagulant) before introducing the phagocytosis object into it, helps to maintain the viability of neutrophils and their functional properties, including responsible for phagocytosis, at a level corresponding to or approaching the level inherent in a given organism in vivo. When storing the blood of the examined organism for more than 30 min, there is a significant violation of the functional properties of neutrophils, which, according to the inventors, is caused by the death of these cells, a violation of the natural processes of their chemotaxis and adhesion, absorption and digestion of microbes by them. Thus, the use during the formulation of the phagocytosis reaction of blood samples of the examined organism, the storage periods of which (before introducing the object of phagocytosis) do not exceed the upper limit of the interval of optimal values of the indicated

parameter, allows to exclude the possibility of violations of the phagocytic function of neutrophils, as well as reduce their viability due to the negative impact of the specified biological factor, which significantly reduces the likelihood of obtaining false and false results of the reaction.

In addition, the claimed conditions and modes of action provide an additional enhancement of the technical result, as well as the achievement of additional advantages in particular cases of the invention. The use of capillaries phagocytosis during the formulation of the reaction, the inner diameter of which is within the range of optimal values (0.2-0.8 mm), determines the optimal parameters of the capillary lumen. Under these conditions, the thickness of the parietal layer of the capillary is sufficient to allow white blood cells to “slip” towards the bottom of the capillary while excluding the possibility of “piercing” the parietal layer by these cells (“piercing” of the parietal layer occurs when capillaries with an inner diameter of more than 0.8 mm). This makes it possible to completely eliminate the contact of leukocytes with the walls of the capillary, which additionally reduces the likelihood of nonphysiological activation of neutrophils due to their adhesion to the walls of the capillary, as well as trauma to these cells on the walls of the capillary. The optimal parameters of the capillary lumen also allow one to obtain a leukocyte sediment, the volume of which is sufficient for the preparation of high-quality smears containing the necessary number of neutrophils for counting (at least 200 cells), which further contributes to the creation of optimal conditions for microscopic evaluation of the reaction results. All this additionally reduces the likelihood of obtaining false and false lower results of the determination of FAN. In addition, the use of capillaries with an optimal inner diameter ensures that there are no technical difficulties when performing the operation “leukocyte isolation” (spontaneous leakage of the contents of the capillary into the external medium is excluded), which makes it possible to obtain a leukocyte suspension of optimal density (when applied to a glass slide). This further reduces the duration of the microscopic evaluation of the reaction results.

Thus, it is the claimed sequence of actions in combination with the modes and conditions for their implementation that eliminates the mechanical, physicochemical, biological and / or immunological factors that negatively affect the viability of peripheral blood neutrophils of the examined organism, the chemotaxis and adhesion of these cells, and also on the processes of absorption and digestion of microbes by them. This creates the conditions for maintaining in vitro the functional properties of the neutrophils of each particular organism responsible for phagocytosis at a level corresponding to or approaching the in vivo level inherent in an individual organism. This, in turn, makes it possible to almost completely exclude the possibility of obtaining false-overestimated and false-underestimated reaction results, thereby providing an increase in the accuracy of determining FAN. Moreover, from the prior art, according to the applicant, the effect of the transformations prescribed by the invention, characterized by significant features distinctive from the prototype, on the achievement of a technical result is not revealed.

The method is as follows. Blood is taken with an anticoagulant in the examined organism. At the same time, blood taken from a finger or from a vein of the examined organism is introduced into a sterile test tube with a preliminarily introduced 5% sodium citrate solution at a 1: 4 ratio of sodium citrate solution. Prepare a mixture for the study of phagocytosis and its processing. At the same time, a microbial suspension, for example, a suspension of staphylococci (which is used, for example, daily agar culture of Staphylococcus epidermidis, strain 9198 by standard), at a concentration of 1 billion standard, is added to a test tube containing blood with an anticoagulant no later than 30 minutes after blood sampling microbial cells in 1 ml (object of phagocytosis) with a ratio of blood with anticoagulant and object of phagocytosis 2: 1. Two capillaries, the inner diameter of which is 0.2-0.8 mm, are filled with the obtained mixture for the study of phagocytosis. One of the ends of each capillary is sealed with a mixture of wax and paraffin and both capillaries are placed in an upright thermostat. Incubate at a temperature of 37 ° C each of their capillaries containing a mixture for the study of phagocytosis, one of the capillaries for 30 minutes, and the other for 120 minutes (2 hours). At the end of the incubation period, each of the capillaries is centrifuged for 10 min at 1500 rpm. After centrifugation is completed, each of the capillaries is disconnected, for example, by filing with a nail file to open the ampoules at the border of the leukocyte sediment and the erythrocyte sediment, followed by breaking of each of the capillaries. A leukocyte pellet is recovered from the corresponding end portion of each of the disconnected capillaries, for example, by transferring a leukocyte pellet from each capillary onto a defatted glass slide (touching the tip of the capillary containing portion of the white blood cell with a tip).

Smears are prepared from a leukocyte pellet extracted from each of the disconnected capillaries. The prepared smears are dried in air, then fixed for 10 min in absolute methyl alcohol and stained with azure-eosin according to Romanovsky-Giemsa. After staining, smears are examined under a microscope in an immersion system: at least 200 cells are counted (total number of neutrophils). The number of neutrophils that have entered into phagocytosis and the total number of microbes absorbed by neutrophils are determined on each smear / hereinafter referred to as "cellular indicators necessary for the subsequent calculation of phagocytosis indices" /. Phagocytosis indices are calculated, which are used as follows:

1. FI determined by the formula (1).

2. PS defined by the formula (2).

Both indicators (FI and PS) are calculated on smears prepared from sediments of 30- and 120-minute incubations (FI30 and FI120, respectively, Ф30 and Ф120).

3. KPF defined by the formula (3).

4. IBN, determined by the formula (4).

The indicators included in formula (4) are the number of microbes killed inside neutrophils (Ч _у ) and the total number of microbes absorbed by neutrophils (Ч _п, Ч _{п ′} ) are estimated, for example, by plating washed and lysed sediment on solid nutrient media (agar) leukocytes according to the technique described in [2, p. 384-386] / hereinafter - "bacteriological indicators necessary for the subsequent calculation of IHD" /. Wherein _y H is determined by the formula:

where H _p - the number of colony forming units / CFU / staphylococcus when plating a lysed sediment of leukocytes incubated for 30 minutes;

H _{P ′} - the number of CFU of staphylococcus when plating a lysed sediment of leukocytes incubated for 120 min;

H _y - the difference between the number of CFU of staphylococcus when plating a lysed precipitate of leukocytes incubated for 30 min and the number of CFU of staphylococcus when plating a lysed precipitate of leukocytes incubated for 120 min.

The obtained values of phagocytosis indicators are compared with normal (for the type of organisms to which the organism being examined) values. When examining patients and healthy people use, for example, the normal values given in [4]. The presence or absence of changes in each of the studied phagocytosis indices is established in comparison with the norm. When finding the values of all the studied indicators of phagocytosis within normal limits, they conclude that there are no violations of the FAN of the examined organism. In the presence of deviations from the norm of at least one of the studied phagocytosis indices, a conclusion is drawn about the presence of violations of the FAN of this organism.

The invention is illustrated by the following examples.

Example 1

Patient Seryozha T., 7 years old. I turned to the outpatient department No. 41 of Children's Children's Hospital No. 1 with complaints of coughing, an increase in body temperature to 38.5 ° C. Sick for 5 days. He received symptomatic therapy (antipyretic and expectorant drugs), but his condition worsened, and therefore he turned to Children's hospital No 1. The patient has a history of recurrent pneumonia, furunculosis, adenoflegmon, osteomyelitis from an early age (from the age of 1 month). The child is also observed by a neurologist about a lag in psychomotor development, nystagmus.

The patient was determined by FAN using the claimed method and the prototype method. During the determination of FAN, 10 ml of blood was taken from a vein from the examined child and introduced into a sterile test tube (initial) with 2.5 ml of 5% sodium citrate solution added in advance. The resulting blood with an anticoagulant was used to set the phagocytosis reaction according to the method of the claimed method and the method of the prototype method.

During the determination of FAN using the inventive method, 0.1 ml was collected from a source tube containing blood with an anticoagulant in a separate (experimental tube). Then, 0 min ml of a suspension of staphylococci (daily agar culture of Staphylococcus epidermidis, pcs. 9198 by standard) at a concentration of 1 billion microbial cells in 1 ml Two capillaries with an inner diameter of 0.8 mm were filled with the obtained mixture for the study of phagocytosis. One of the ends of each capillary was sealed with a mixture of wax and paraffin and both capillaries were placed in an upright thermostat. Each of the capillaries containing a mixture for the study of phagocytosis was incubated at a temperature of 37 ° C: one of the capillaries for 30 min and the other for 120 min (2 h). At the end of the incubation period, each of the capillaries was centrifuged for 10 min at 1500 rpm. After centrifugation was completed, each of the capillaries was separated by sawing with a nail file to open the ampoules at the border of the leukocyte sediment and erythrocyte sediment, followed by breaking of each of the capillaries. The leukocyte pellet was removed from the corresponding end portion of each of the disconnected capillaries by transferring the leukocyte pellet from each capillary onto a defatted glass slide (touching the tip of the capillary containing leukocyte tip to the glass slide).

Smears were prepared from a leukocyte pellet extracted from each of the disconnected capillaries. The prepared smears were dried in air, then fixed for 10 min in absolute methyl alcohol and stained with azure-eosin according to Romanovsky-Giemsa. After staining, each of the smears was examined under a microscope in an immersion system: 200 cells were counted (total number of neutrophils). We determined on each of the smears the cellular indices necessary for the subsequent calculation of phagocytosis indices:

The phagocytosis indices were calculated: PI - according to the formula (1), PS - according to the formula (2), CPF - according to the formula (3):

We determined the bacteriological indicators necessary for the subsequent calculation of IHD by plating on washed nutrient media (agar) the washed and lysed sediment of leukocytes incubated for 30 min and 120 min (according to the method [2]):

Calculated the number of dead within neutrophils microbes (H _y) as the difference between the number of CFU Staphylococcus during sowing lysed sludge leukocytes incubated for 30 min, and the number of CFU Staphylococcus during sowing lysed sludge leukocytes incubated for 120 min, the formula (5):

H _y = 156-101 = 55 CFU of staphylococcus

Calculated the index of IHD according to the formula (4):

The results of the definitions and calculations are presented in the summary table.4.

When comparing the obtained values of phagocytosis indices with the normal values given in [4], it was found that: FI30, FI120, PS30, PS120 and IBN are significantly reduced (the values of these indicators are outside the lower boundary of the intervals of the corresponding normal values); KFCH - within normal limits. Thus, the results obtained using the claimed method showed the presence of a defect in the phagocytic immunity link, namely, the presence of violations of both the initial and final stages of the phagocytosis process. The time spent on the determination procedure amounted (according to the results of timekeeping of the authors of the proposed method) to 146 minutes (2.4 hours).

In parallel, the patient was determined FAN according to the method of the prototype method [4]. In this case, the blood remaining in the original tube with the anticoagulant was mixed, after which it was centrifuged for 10 min at 1000 rpm. Plasma with a layer of white blood cells was pipetted, placed in a clean centrifuge tube and 10 ml of 199 medium was added. WBCs were washed twice in medium 199 by centrifugation twice for 10 min at 1000 rpm. After the last centrifugation, the supernatant and part of the cell suspension were pipetted out, leaving 0.2 ml of the cell suspension in medium 199 in a centrifuge tube. Then, 0.3 ml of staphylococcus suspension was added to the tube containing 0.2 ml of white blood cells in medium 199 (culture the same as in the determination of FAN by the claimed method) at a concentration of 1 billion microbial cells in 1 ml, after which 0.15 ml of a pool of fresh donor serum of group AB (IV) was added (time from the moment of blood sampling from the examined child to the introduction of the object of phagocytosis in leukocyte suspension amounted, according to the timing of the inventors, 43 min). The components were mixed and the resulting mixture was thermostated to study phagocytosis for 30 min at a temperature of 37 ° C. After this time, 5 ml of isotonic sodium chloride solution heated to 37 ° C was added to the test tube, shaken and centrifuged for 10 min at 1500 rpm. At the end of centrifugation, the supernatant was removed and smears were made from leukocyte suspension, the remains of which were placed in a thermostat at 37 ° C to continue incubation for another 90 min (total incubation time 120 min). Then the smears were prepared again from the sediment of a 120-minute incubation. Smears from leukocyte sediments (after a 30-minute and 120-minute incubation) were prepared on fat-free glass slides. Fixation and staining of smears was performed in the same way as in the determination of FAN by the claimed method. On each of the smears, the cellular indices necessary for the subsequent calculation of the phagocytosis indices were determined, and the phagocytosis indices were calculated: FI, PS, KPh - similarly to the claimed method. We determined the bacteriological parameters necessary for the subsequent calculation of IHD, and calculated IHD similarly to the claimed method.

The results of the determinations and calculations are given in the summary table.4.

A comparison of the results of the determination with normal values [4] showed that the obtained values of all the studied phagocytosis indices were within the ranges of the corresponding normal values. Thus, the prototype method did not reveal violations of the FAN. The duration of the determination procedure, according to the timing of the inventors, was 198 min (3.2 hours).

A comprehensive examination of the patient. Clinical examination revealed albinism, silver glow of hair when illuminated by an electric lamp, and the absence of pigment deposition in the retina. An x-ray of the chest revealed infiltration in the lower lobe of the right lung.

Laboratory test results

In a blood test revealed neutropenia:

Hb 116 g / l; Er 3.8 · 10 ¹² / l; white blood cells 5.6 · 10 ⁹ %; platelets 180 · 10 ¹² / l; stab 7%; segmented 8%; eosinophils 4%; lymphocytes 70%; basophils 2%; monocytes 9%; ESR 25 mm / h.

The immunogram revealed:

1) a slight decrease in the relative number of T-lymphocytes, a slight increase in the number of B-lymphocytes:

CD3 48% (norm 50-76%);

CD4 35% (norm 31-46%);

CD8 28% (normal 26-40%);

CD16 15% (norm 9-16%);

CD20 18% (norm 11-16%);

CD25 25% (normal 13-24%);

2) a slight increase in the concentration of immunoglobulin M (Ig M):

Ig G 690 mg% (norm 583-1783 mg%);

Ig M 150 mg% (normal 24-112 mg%);

Ig A 95 mg% (normal 85-559 mg%);

3) the content of the components of the complement system corresponded to the norm:

C1q 130 μg / ml (normal 100-250 μg / ml);

C3 850 μg / ml (norm 700-1800 μg / ml);

C3a 0.07 μg / ml (normal 0.05-0.15 μg / ml);

C4 400 mcg / ml (normal 200-500 mcg / ml);

C5a 0.025 μg / ml (normal 0.01-0.03 μg / ml);

4) spontaneous and induced production of cytokines slightly differed from the norm:

5) the level of circulating immune complexes / CEC / corresponded to the norm:

112 units / ml (normal up to 120 units / ml);

6) a significant reduction in chemotaxis of neutrophils:

migration index under the influence of FMLP 0.2 (norm 2.6-2.8);

7) a slight decrease in the test with nitro blue tetrazolium:

granules of formazan were detected in 8% of neutrophils (norm 11-40%).

Taking into account the anamnesis, clinical presentation and a comprehensive laboratory examination, the diagnosis was made: Chediak-Higashi syndrome. Given that Chediak-Higashi syndrome according to the work [7] refers to congenital immunodeficiencies accompanied by defects in phagocytosis, the results of a comprehensive examination confirmed the results of the determination of FAN obtained using the claimed method, and allowed us to evaluate the results of determining FAN by the prototype method as false-raised.

Example 2

Patient Tatyana S., 14 years old. I turned to the outpatient department No. 41 of Children's Children's Hospital No. 1 with complaints of pustular skin rashes, an increase in body temperature to 37.8 ° C. Deterioration was noted two weeks ago: pustular skin rashes (boils) appeared in various parts of the body (right shoulder, abdominal wall, thighs). The girl received treatment in the district clinic (dressings with furatsilinom, various ointments, opening of boils), but boils appeared in new areas of the body. A patient with a history of 1.5 years had recurrent furunculosis with exacerbations in the autumn-winter season.

The patient was determined by FAN using the claimed method and the prototype method analogously to example 1.

When determining the FAN using the claimed method, capillaries with an inner diameter of 0.6 mm were used. The time from the moment of blood sampling from the examined child to the introduction of the object of phagocytosis into the blood with the anticoagulant was 16 minutes.

The results of the determinations and calculations are given in the summary table.5.

Comparison of the obtained values of phagocytosis indices with normal values revealed a significant decrease in the frequency of 30, phase 120 and IHB with a slight decrease in FI30, FI120 (KPh slightly increased), which indicated the presence of a defect in the phagocytic immunity, namely, the presence of violations of both the initial and the final stages of the phagocytosis process. The determination procedure took 152 minutes (2.5 hours).

In parallel, the patient was determined by FAN using the prototype method. The time from the moment of blood sampling from the examined child to the introduction of the phagocytosis object into the leukocyte suspension was 45 minutes.

The results of the determinations and calculations are given in the summary table.5.

The results of the comparison of the values of phagocytosis indices obtained using the prototype method with normal values turned out to be similar to the corresponding results of the comparison of the data obtained using the claimed method (a significant decrease in PS30, PS120 and IBN, a slight decrease in PI30, FI120, an increase in KPH). This allowed us to draw conclusions similar to the conclusions of the claimed method (the presence of a defect in the phagocytic immunity; the presence of violations of both the initial and final stages of the phagocytosis process). The determination procedure took 198 min (3.3 h).

A comprehensive examination of the patient was carried out analogously to example 1. During a clinical examination revealed the presence of boils on the right thigh, on the right lower leg, left forearm; body temperature 37 ° C. The physical development of the child was age appropriate. Repeated examination of blood sugar and sugar curves revealed no abnormalities.

Laboratory test results

A blood test revealed leukocytosis with a neutrophilic left shift: Нb 135 g / l; Er 4.1 · 10 ¹² / l; white blood cells 15.7 · 10 ⁹ / l; platelets 220 · 10 ¹² / l; young 3%; stab core 15%; segmented 48%; eosinophils 2%; lymphocytes 25%; monocytes 7%; ESR 32 mm / h.

The immunogram revealed:

1) a slight increase in the number of b-lymphocytes:

CD3 56%;

CD4 34%;

CD8 27%;

CD16 12%;

CD20 19%;

CD25 25%;

2) the concentration of immunoglobulins corresponded to the age norm:

Ig G 840 mg% (norm 575-1607 mg%);

Ig M 130 mg% (normal 26-135 mg%);

Ig A 230 mg% (norm 86-588 mg%);

3) the content of the components of the complement system corresponded to the norm:

C1q 210 μg / ml;

C3 1200 μg / ml;

C3a 0.12 μg / ml;

C4 450 μg / ml;

C5a 0.018 μg / ml;

4) spontaneous and induced production of cytokines slightly differed from the norm:

5) the level of the CEC corresponded to the norm:

118 units / ml;

6) chemotaxis of neutrophils corresponded to the norm:

migration index under the influence of FMLP 2.6;

7) a decrease in the test with nitro blue tetrazolium:

Formazan granules were detected in 6% of neutrophils.

Comprehensive examination data allowed us to conclude that the cause of recurrent furunculosis in a child is immunodeficiency - a defect in phagocytosis, since deviations from the norm were not found in assessing the remaining parts of the immune system. Thus, in this case, there was a coincidence of the results of the determination of FAN using the claimed method and the prototype method. Moreover, the results of the phagocytic reaction correlated with the data of a comprehensive examination.

Example 3

Child Nikolai P., 16 years old. It was examined in the outpatient department No. 41 of Children's Children's Hospital No. 1 in the medical examination order. Complaints during the inspection did not show. A history of rare (1-2 times a year) SARS.

The boy was determined FAN using the claimed method and the prototype method analogously to example 1.

During the determination of FAN according to the method of the claimed method, capillaries with an inner diameter of 0.2 mm were used. The time from the moment of taking blood from the examined child to the introduction of the object of phagocytosis into the blood with an anticoagulant was 5 minutes.

The results of the determinations and calculations are given in the summary table.6.

When comparing the obtained values of phagocytosis indices with normal values of deviations from the norm, it was not revealed, which allowed us to conclude that there were no violations in the phagocytosis system in the examined child. The duration of the determination procedure is 136 minutes (2.3 hours).

In parallel, the boy was determined FAN according to the prototype method. The time from the moment of blood sampling from the examined child to the introduction of the phagocytosis object into the leukocyte suspension was 36 minutes.

The results of the definitions and calculations are presented in the summary table.6.

As a result of comparing the data obtained using the prototype method with normal values, a significant decrease in PS30, PS120 and IHD was revealed, on the basis of which it was possible to conclude that the child has serious disorders in the phagocytosis system (at the initial and final stages of the phagocytosis process ) The duration of the determination procedure is 178 minutes (3.0 hours).

A comprehensive examination of the child. Clinical examination revealed flat feet. Deviations from other organs and systems were not found.

Laboratory examination results:

In the blood test, deviations from the norm were not detected:

Hb 145 g / l; Er 4.5 · 10 ¹² / l; white blood cells 6.3 · 10 ⁹ %; platelets 250 · 10 ¹² / l; stab 3%; segmented 53%; eosinophils 2%; lymphocytes 37%; monocytes 5%; ESR 3 mm / h. In the immunogram:

1) the content of T and B lymphocytes corresponded to the norm:

CD3 40%;

CD4 43%;

CD8 32%;

CD16 15%;

CD20 12%;

CD25 21%;

2) the concentration of immunoglobulins corresponded to the age norm:

Ig G 1250 mg% (norm 770-1510 mg%);

Ig M 112 mg% (norm 67-208 mg%);

Ig A 185 mg% (normal 134-297 mg%);

3) the content of the components of the complement system corresponded to the norm:

C1q 210 μg / ml;

C3 950 μg / ml;

C3a 0.11 μg / ml;

C4 410 mcg / ml;

C5a 0.015 μg / ml;

4) spontaneous and induced production of cytokines corresponded to the norm:

5) the level of the CEC corresponded to the norm:

96 units / ml;

6) chemotaxis of neutrophils corresponded to the norm:

migration index under the influence of FMLP 2.7;

7) the test with nitro blue tetrazolium corresponded to the norm:

Formazan granules were detected in 38% of neutrophils.

Comprehensive survey data showed the following. An orthopedic pathology was discovered in a child: flat feet. Diseases that would indicate abnormalities in the immune system are not noted. In particular, no diseases were identified that could cause a decrease in phagocytosis. There were also no medical, diagnostic and / or preventive procedures that could cause a decrease in phagocytosis. Thus, the results of a comprehensive examination, which indicated the absence of violations of the FAN, confirmed the determination results obtained using the claimed method, and made it possible to evaluate the results of determining the FAN by the prototype method as false.

The claimed method was used in the examination of 342 children aged 3 to 15 years, which was carried out on the basis of Children's hospital No 1 (in the outpatient department No 41). For comparison, the determination of FAN was carried out using the prototype method [4].

All children underwent a comprehensive examination using various clinical, instrumental and laboratory methods, including immunological methods (determination of the number of T and B lymphocytes; concentration of immunoglobulins A, M, G; content of components of the complement system; determination of spontaneous and induced production of cytokines; determination of the level of the CEC; determination of the FAN, etc.). The determination of FAN during a comprehensive examination was carried out by studying the chemotaxis of peripheral blood leukocytes according to the method described in [2], and by setting up a test with nitro blue tetrazolium according to the method described in [3]. Taking into account the anamnesis, clinical presentation and comprehensive examination, clinical diagnoses were made (hereinafter referred to as “the results of the comprehensive control examination / CCW /”). At the same time, the examined children were divided into the following groups:

Group I - patients with Chediak-Higashi syndrome (2 people);

Group II - patients with recurrent furunculosis (12 people);

Group III - patients with recurrent lymphadenitis (6 people);

Group IV - patients with recurrent otitis media, sinusitis, pneumonia (11 people);

Group V - patients with chronic gastritis, duodenitis, biliary dyskinesia (112 people);

Group VI - patients with congenital heart defects (48 people);

VII group - patients with disorders of the musculoskeletal system (scoliosis, flatfoot, dysplasia of the hip joint) (136 people);

VIII group - practically healthy children (15 people).

Statistical processing of the survey results was carried out using the Fisher and Wilcoxon-Mann-Whitney criteria [6].

The results are presented in tables 7, 8. Table 7 gives a comparative analysis of the accuracy of determining FAN in children with various diseases (in groups) and practically healthy children using the claimed method and the prototype method. In the table. 8 shows the summary data characterizing the effectiveness of the claimed method and the prototype method.

From table 7 it is seen that the violations of the FAN were confirmed using CLC in 22 children (in 2 patients with Chediak-Higashi syndrome (group I); in 12 patients with recurrent furunculosis (group II); in 5 patients with recurrent lymphadenitis (III group); in 3 patients with recurrent otitis media, sinusitis, pneumonia (group IV)).

When using the claimed method, violations of the FAN were detected in all these patients. However, the absence of violations of the FAN, established using the proposed method, in all cases (320 children) fully correlated with the results of the CCW (in 1 patient of group III; in 8 patients of group IV, as well as in all patients of groups V-VII and practically healthy children (VIII group)).

Using the prototype method, abnormalities in the phagocytosis system (confirmed by KCO) were revealed in 15 patients (in 1 patient with Chediak-Higashi syndrome (I gr.); In 9 patients with recurrent furunculosis (II gr.); In 3 patients with recurrent lymphadenitis (III gr.) and in 2 patients with recurrent otitis media, sinusitis, pneumonia (IV gr.)). In 7 patients with confirmed KCO violations of the FAN (in 1 patient with Chediak-Higashi syndrome (I gr.); 3 patients with recurrent furunculosis (II gr.); 2 patients with recurrent lymphadenitis (III gr.) And 1 patient with recurrent otitis media (IV gr.)) the values of phagocytosis indices obtained using the prototype method corresponded to normal values (false-raised determination results). In addition, the violations of the FAN identified using the prototype method in 43 cases were not confirmed by the results of the CCL (in 15 patients with chronic gastritis, duodenitis, biliary dyskinesia (V gr.): In 11 cases, false-determined results, in 4- x cases - false low; in 3 patients with congenital heart defects (VI gr.): in 2 cases - false high results, in one case false low; in 21 children with disorders of the musculoskeletal system (group VII): in 16 ty cases - false-overestimated results, in 5 cases - ozhnozanizhennye and in 4 healthy children (VIII c.) in 3 cases - lozhnozavyshennye results in one case - lozhnozanizhenny).

As follows from the data of table 8, the claimed method provides for its implementation 100% accuracy of determination of FAN, which is 14.6% higher than the accuracy of determination using the prototype method. At the same time, with the violations of the FAN confirmed by KCO, the results of the determination by the prototype method were considered erroneous in 31.8% of cases, and in the absence of violations in the phagocytosis system (confirmed by KCO) in 13.4% of cases (Table 7). In addition, the claimed method, as can be seen from the table. 8, allows to reduce the duration of the procedure for determining FAN by an average of 1.3 times compared with the prototype method (by eliminating the need for preliminary separation of leukocytes and erythrocytes and the isolation of leukocytes as special operations during the preparation of the mixture for the study of phagocytosis, the separation of leukocytes and red blood cells directly during the reaction of phagocytosis and simplification of the operation "leukocyte isolation"). The duration of the microscopic evaluation of the reaction results is comparable with the same indicator of the prototype method. Thus, the claimed method, when implemented, provides high accuracy in determining the FAN with the relative simplicity of technical performance and relatively short analysis time.

Sources of information

1. Chernushenko E.F., Kogosova L.S. Immunology and immunopathology of lung diseases. - Kiev: Health, 1981. - P.169.

2. Immunological methods: Trans. with him. / Ed. G. Fremel. - M .: Medicine, 1987. - S.333-338, 384-386.

3. Douglas S.D., Cui P.G. The study of phagocytosis in clinical practice: Trans. from English - M .: Medicine, 1983. - S.63-64, 87-88.

4. Laboratory research methods in the clinic: Handbook / V.V. Menshikov, L. N. Delectors, R. P. Zolotnitskaya and others. Ed. V.V. Menshikova. - M .: Medicine, 1987. - S.310-311 (prototype).

5. Lymphocytes: Methods: Trans. from English / Ed. J. Claus. - M .: Mir, 1990. - P.29.50.

6. Gubler EV Computational methods of analysis and recognition of pathological processes. - L .: Medicine, 1978. - S.72-75, 84-91.

7. Nelson textbook of pediatrics. - 15th ed. / edited by R.E. Behrman, R.M. Kliegman, A.M. Arvin; senior editor W.E. Nelson. - W. B. Saunders Company, 1996. - P.561-609.

8. Lurie A.A. Chromatographic Materials: Reference. - M .: Chemistry, 1978. - S.190-191.

9. Pediatric Hematology: A Guide for Physicians. / Ed. N.A. Alekseeva. - St. Petersburg: Hippocrates, 1998 .-- S.97-105.

Claims (2)

1. A method for determining the phagocytic activity of peripheral blood neutrophils, including blood sampling with an anticoagulant, preparation of a mixture for the study of phagocytosis containing leukocytes and the object of phagocytosis, isolation of leukocytes, processing of the mixture for the study of phagocytosis by incubation and centrifugation, preparation of smears from precipitation obtained by centrifugation, followed by determination on each of the smears, the number of neutrophils that have entered into phacitosis, and the total number of microbes absorbed by neutrophils, calculation of indicators phagocytosis and comparison of the obtained values of phagocytosis indices with normal values, characterized in that during the preparation of the mixture for the study of phagocytosis, the object of phagocytosis is first introduced into the blood with the anticoagulant no later than 30 minutes after its collection, after which two capillaries are filled with the obtained mixture for the study of phagocytosis treatment of the mixture is carried out by incubation at a temperature of 37 ° C for each of the capillaries, one of the capillaries being incubated for 30 minutes and the other for 2 hours, and centrifuged each of these capillaries for 10 min at 1500 rpm, the selection of leukocytes is carried out immediately after centrifugation by separating each of the capillaries at the border of the leukocyte sediment and erythrocyte sediment and extracting the leukocyte sediment from the corresponding part of each of the disconnected capillaries, smear preparation is carried out from the sediment leukocytes, and as indicators of phagocytosis, the phagocytic index, phagocytic number, phagocytic number coefficient and bactericidal index n ytrofilov. 2. The method according to claim 1, characterized in that during the preparation of the mixture for the study of phagocytosis and its processing, capillaries with an inner diameter of 0.2-0.8 mm are used.