The method of determination of phagocytic activity of neutrophils in the peripheral blood of living organisms

 

(57) Abstract:

The invention relates to medicine, namely to immunology, Allergology, pulmonology, dermatology, infectious Microbiology, and veterinary medicine. Essence: create a way to exclude the possibility of obtaining linesavannah and loosening results of the determination through the creation of conditions for preservation in vitro functional properties of neutrophils of each individual organism. This produces the blood with the anticoagulant of the examined body. Carry out the preparation of a mixture for the study of phagocytosis containing leukocytes and the object of phagocytosis, in which first make the object of phagocytosis in blood with anticoagulant no later than 30 minutes after its intake, then fill the mixture two capillary. They process the mixture for the study of phagocytosis by incubation at a temperature of 37°With each of the capillaries, and one of capillaries incubated for 30 min and the other for 2 h, and centrifugation of each of the capillary for 10 min at 1500 rpm Directly after centrifugation to produce a selection of cells by separation of kaidou from the corresponding part of each of the disconnected capillaries. Prepare smears from the precipitation of cells obtained after centrifugation. Define each of the strokes number of neutrophils, which came in phagocytosis, and the total number of absorbed neuropile microbes. Expect phagocytosis indices, which are used: phagocytic index, phagocytic number, the coefficient of phagocytic number and index bactericides neutrophils. Compare the obtained values of phagocytosis with normal values and comparing conclude the presence or absence of disorders of phagocytic activity of neutrophils analyzed peripheral blood. The technical result - expanding Arsenal of ways to assess the phagocytic activity of the blood. 1 C.p. f-crystals, 8 PL.

The invention relates to medicine, namely to immunology, Allergology, pulmonology, dermatology, infectious Microbiology, veterinary medicine, and can be used in the evaluation of the immune status of healthy and diseased organism.

There is a method of determining the phagocytic activity of leukocytes from patients and healthy individuals [1]. The setting reaction of phagocytosis according to the method similar as follows. In the course prepared the analyzed blood and 0.1 ml of a suspension of the test microbe, which is used as a laboratory strain of Staphylococcus No. 209 or Century mesenthericus (daily agar culture according to standard) at a concentration of 400 million microbial cells in 1 ml (object phagocytosis). All components are thoroughly mixed, after which they process the mixture for the study of phagocytosis. While the test tube is placed for 30 min in a thermostat (37°C). After incubation, the test tube with the mixture centrifuged for 3 min at 1000 rpm and Then from the upper sediment layer is prepared brushstrokes that capture the mixture Nikiforova (equal parts of alcohol and ether) and stained by Romanovsky-Giemsa. Under the microscope looking at 100 leukocytes (total WBC) and count the total number of absorbed by leukocytes microbes. Calculate indicators of phagocytosis, which use 2 indicators:

1. The percentage faguoqitirute of leukocytes, i.e., the number of cells 100, showed phagocytic activity (this figure corresponds to the meaning of the indicator of phagocytic index", the definition of which is introduced below in the description of the prototype method).

2. Phagocytic number, i.e. the number of microbes, absorbed on average, one leukocyte.

For healthy individuals por the exhaust gas [1] does not ensure the achievement of the technical result of the claimed method in connection with the following. Provided by technique of the method-analogue time setting mode incubation mixture for the study of phagocytosis (30 min) allows you to define only two indicators of phagocytosis: phagocytic index (after 30-minute incubation) and phagocytic number (after 30-minute incubation). These figures reflect the adsorption capacity of neutrophils in the early stages of phagocytosis. The lack of research final stages of phagocytosis (e.g., after 2-hour incubation) leads to the impossibility to determine such important indicators of phagocytosis, as the ratio of phagocytic number and index bactericides neutrophils (for calculation which are used, in particular, the parameters determined on smears prepared from precipitation 2-hour incubation). The coefficient of phagocytic number reflects the absorption capacity of neutrophils in the final stages of phagocytosis, and the index of bactericides neutrophil ability phagocyte to digest the captured microbes in the final stages of phagocytosis. Both figures are included in the set of phagocytosis indices, each of which is necessary and together sufficient to ensure the adequacy of ozenc to be discrete and to cover comprehensively, and isolated at different stages of phagocytosis (chemotaxis, adhesion to the microbe, absorption, digestion of microbes). In this regard, the lack of metrics that reflect the process of phagocytosis in the dynamics, minimizes in a number of instances, the likelihood of detection in the system of phagocytosis. In addition, the inability to assess bactericidal neutrophils will not allow to detect the disease with predominant disturbance bactericides (such as syndrome Chediak-Higashi and chronic Wegener), as well as diseases in which violations of phagocytosis (including violations of bactericides) can play a leading pathogenetic role (pyoderma; adenoidectomy; abstemious pneumonia; nonhealing wounds; postoperative complications; secondary immunodeficiencies caused by drug therapy and so on). Evaluation of the reaction of phagocytosis in these patients is necessary for the choice of tactics of treatment, because the same clinical manifestations can be caused by disturbances in other parts of the immune system (such as system of a compliment, antibody). In this regard, the technique of the method-analogue of [1] is fundamentally unsuitable for the evaluation of phagocytic activity of neutrophils in a wide range of illness is no pre-separation of leukocytes and erythrocytes, and selection of cells as special operations during the mixture preparation for the study of phagocytosis. Separation of leukocytes and erythrocytes are produced in the processing phase mixture for the study of phagocytosis by centrifugation of this mixture (i.e., after termination of the reaction phagocytosis at the initial stage). Used in the method-analogue mode centrifugation (3 min at 1000 rpm) does not provide a complete deposition of cells, a significant part of them remains in the supernatant. Thus, the experiments of the inventors have shown that the dependence of the number of cells in the supernatant from the centrifugation time (at 1000 rpm) as follows:

According to the method similar selection of cells produced after centrifugation of the mixture for the study of phagocytosis by sucking leukocyte film on the layer of red blood cells. When a short centrifugation (3 min) the film formed primarily neutrophils, absorbing microbes (i.e., cells with more weight), and neutrophils that have not entered in phagocytosis (with less weight), can remain in the supernatant. All this significantly reduces the accuracy of determination of phagocytic activity of leukocytes. In addition, the separation of leukocytes from erythrocytes by sucking it does not provide what you authors of the proposed method showed in spite of this, the production reaction of phagocytosis admixture of blood smears can be up to 360 erythrocytes for 1 leukocyte). This greatly complicates the microscopic evaluation of the results of the reaction, thereby increasing its duration. High probability of injury of cells on the bottom and wall of the tube (under stirring of the mixture for the study of 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 that are responsible for phagocytosis, which also reduces the accuracy of the analysis.

Thus, features performances reaction of phagocytosis using a method similar to [1] determine the limited scope of the methodology (due to the fundamental impossibility of an adequate assessment of the results of reactions in a number of diseases), as well as the relatively low accuracy of determination of phagocytic activity of leukocytes in cases where the assessment of phagocytosis using a method similar to fundamentally possible.

In modern basic guidelines immunological methods [2; 3] is recommended for a wide laboratory practice primary, and final stages of the reaction. In addition, these guidelines generally provide for initial separation of erythrocytes and leukocytes, followed by separation of leukocytes as a special operation in the course of preparation of the mixture for the study of phagocytosis.

Closest to the claimed solution of the essential features is the way to determine the phagocytic activity of neutrophils /FAN/ peripheral blood of patients and healthy individuals [4]. The method adopted for the prototype includes the following stages. Produce blood with anticoagulant in a patient patient. In a sterile tube with a pre-made solution of 0.6 ml of heparin, diluted 1:10, make 10 ml of blood from the cubital vein. Carry out the preparation of a mixture for the study of phagocytosis. First make a separation of leukocytes and erythrocytes, followed by separation of leukocytes. During this operation, the blood is thoroughly mixed and centrifuged 10 min at 1000 rpm Plasma together with a layer of cells gently sucked off and placed in a clean centrifuge tube, adding 5-6 ml of medium 199. Leukocytes washed by centrifugation for 10 min at 1000 rpm Nagoshi procedure soft centrifugation. After the last centrifugation pipette the supernatant is sucked off and part of the cell suspension, leaving the centrifuge tube 0.2 ml of cell suspension in medium 199. Then in a test tube containing 0.2 ml of cells in medium 199, contribute 0.3 ml of microbial suspension, which uses 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 (object phagocytosis), and then add 0.15 ml of a pool of fresh donor sera AB (IV) group (optionsarray factor). Careful rodirovanie mix the components, and then make processing of the mixture for the study of phagocytosis. During this stage the mixture thermostatic 30 min at 37°C. Upon expiration of the time specified in a test tube add 5 ml of warmed up to 37°With isotonic sodium chloride solution, shaken and centrifuged 10 min at 1500 rpm after centrifugation the supernatant is removed and make the strokes of the leukocyte suspension, the remains of which are again placed in a thermostat at 37°to continue incubation for 90 min (total incubation time 120 min). Then smears prepared again from the sediment ducasa otomat low fat slides. Prepared smears air-dried, then fixed for 10 min in absolute methyl alcohol and paint Romanovsky-Giemsa Azur-eosin. After staining smears are viewing under the microscope in the immersion system: consider not less than 200 cells (total number of neutrophils). Define each of the strokes number of neutrophils, which came in phagocytosis, and the total number of absorbed Klebsiella microbes. Calculate indicators of phagocytosis, which use the following:

1. The phagocytic index of /PHI/, defined by the formula:

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

Both indicators (PHI and FC) rely on smears made after the 30-minute and 120-minute incubation (respectively FI and FI; PC and FC).

3. The coefficient of phagocytic number (CFC) defined by the formula:

4. Index bactericides neutrophils /BIN/ defined by the formula:

where IBN-index bactericides neutrophils, %,

Hy- the number of people killed inside Klebsiella microbes;

Hp- total number of tx2">

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

The matching results establish the presence or absence of change of each of the studied parameters of phagocytosis compared to the norm. When the values of all the studied parameters of phagocytosis in normal limits make the conclusion about the absence of violations FAN of the examined patient. If there are deviations from the norm at least one of the studied parameters of phagocytosis conclude that the violations of the FAN in a given patient.

Prototype method [1] does not allow to obtain a technical result achieved when using the inventive method, for the following reasons. Provided by the method of the prototype method initial separation of leukocytes and erythrocytes and release of white blood cells, on the one hand, provides a relatively high purity selection of cells, contributing to the creation of optimal conditions for the microscopic evaluation of the results of the reaction. However, on the other hand, a preliminary separation of leukocytes and erythrocytes eliminates the possibility of the formation during zentrifugen tubes. The procedure of the preliminary separation of the formed elements of the blood itself is interfaced with multiple centrifugation (centrifugation plasma with leukocytes in the preparation of leukocyte suspension, repeated centrifuging the suspension of leukocytes in the money laundering). 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 pre-separation of leukocytes and erythrocytes, and at the stage of processing the mixture), in the absence of erythrocyte pillows all leukocytes in contact with the tube wall, falling on her bottom. The trauma of cells on the bottom and wall of the tube during centrifugation may lead to violations of their functional properties varying severity (including properties, responsible for phagocytosis), and death of these cells. In particular, can be neesiana activation of a significant number of leukocytes due to their adhesion to the bottom and to the walls of the tubes. The presence in the method-prototype multiple mixing a mixture containing leukocytes, as a special operation (during pre-separation uniform is the query result involuntary shaking of the test tube when manipulating the analysis (by placing the tubes into thermostat and remove it from thermostat, when placing the tubes in a centrifuge and removing it from the centrifuge, when transferring tubes from place to place, etc.,) it also significantly increases the possibility of traumatic contact of leukocytes with the walls and bottom of the tube. All this greatly increases the probability of obtaining linesavannah and loosening the results, significantly reducing the accuracy of the FAN.

Preliminary separation of leukocytes and erythrocytes, which results in the destruction of autologous plasma, leads to the destruction contained in the plasma autologous factors. This necessitates making the mixture for the study of phagocytosis opsonizing factor - donor serum, which, along with factors that enhance phagocytosis, may contain cytotoxic factors, damaging the cells. The result may change the functional properties of cells (including may be the suppression or activation of phagocytosis) and to decrease their viability. This may also contribute to the acquisition linesavannah and loosening results of the reaction and reduce the accuracy of the FAN.

Object of the invention is to provide a method for determining fagozyt the teachings linesavannah and loosening results of the determination through the creation of conditions for preservation in vitro functional properties of neutrophils of each individual organism, responsible for phagocytosis, as well as their viability at a level that meets or approaching individually specific to the organism level in vivo, by eliminating mechanical physico-chemical, biological and/or immunological factors that have a negative impact on the processes of chemotaxis and adhesion of neutrophils on the processes of absorption and digestion of microbes, as well as causing the death of these cells.

The problem is solved in that in the method of determining the FAN peripheral blood of living organisms, including blood with anticoagulant; preparation of a mixture for the study of phagocytosis containing leukocytes and the object of phagocytosis, including the selection of cells; processing the mixture for the study of phagocytosis, including its incubation for 2 h at a temperature of 37°C and centrifugation after 30 min of incubation and after 2 h incubation for 10 min at 1500 rpm; preparation of smears from obtained after centrifugation precipitation thirty-minute and two-hour incubation with their subsequent fixation and staining; the definition of each of the strokes in the number of neutrophils, which came in phagocytosis, and the total number of absorbed neutrophils minimi values, the results of which make the conclusion about the presence or absence of violations FAN studied peripheral blood, according to the invention during the preparation of the mixture for the study of phagocytosis first make the object of phagocytosis in blood with anticoagulant no later than 30 minutes after its intake, then fill the mixture for the study of phagocytosis two capillary. Processing the mixture to study phagocytosis is performed by incubation at a temperature of 37°With each of the capillaries containing this mixture, one of capillaries incubated for 30 min and the other for 2 h, and centrifugation of each of the capillary for 10 min at 1500 rpm Allocation of leukocytes produce directly after centrifugation by the end of each of the capillaries on the border of the host sediment leukocytes and sediment erythrocytes and sediment extraction of cells from the corresponding part of each of the disconnected capillaries. Preparation of smears carried out precipitation of leukocytes obtained from each of the disconnected capillaries. As indicators of phagocytosis using phagocytic index, phagocytic number, the coefficient of phagocytic number and index sledovanie phagocytosis and processing of capillaries with an inner diameter of 0.2 to 0.8 mm

Selection and justification of the optimal characteristics of causal important parameters of the proposed method (i.e., parameters that affect the achievement of the technical result) was carried out as follows. As a cause-significant parameters were considered:

the time from the moment of drawing blood from a living organism to make the object of phagocytosis in blood with anticoagulant (quantitative parameter, reflecting the implementation of actions);

modes and conditions of implementation of the processing of the mixture for the study of phagocytosis and subsequent selection of cells as the main factor that enables the separation of leukocytes and erythrocytes directly during the reaction of phagocytosis, but also as a factor in the optimization of complex actions in terms of preservation of the functional properties of neutrophils, including those responsible for phagocytosis, and the viability of these cells.

The choice of the optimal time from the time of collection of blood from a living organism to make the object of phagocytosis in blood with anticoagulant was carried out by varying the time parameter on the background of the optimum characteristics of other parameters of the proposed aggregate (i.e., when the optimal mode is their leukocytes"). With 20 healthy children aged 3 to 15 years took 0.6 ml of blood from a finger and made in a test tube (the original) with a pre-made 5% solution of sodium citrate (0.15 ml per tube). From each source tube (containing blood one child) were collected 6 samples of blood with anticoagulant (0.1 ml) in separate test tubes (6 experimental tubes). In each of the experimental tubes containing the blood sample with anticoagulant, contributed to 0.05 ml of a suspension of Staphylococcus (daily agar culture of Staphylococcus epidermidis pieces 9198 according to the standard) at a concentration of 1 billion microbial cells in 1 ml (object phagocytosis) at various times from the time of collection of blood in the subject of the child: in the 1st, 2nd, 3rd, 4th, 5 th and 6-th pilot tubes, respectively, after 5, 15, 30, 45, 60 and 120 min after blood collection. The lower bound (5 min) of the studied interval, determined by the results of the timing of the authors of the invention, represented the minimum time required for delivery blood of the child from the treatment room to the laboratory and subsequent sampling of blood with anticoagulant from the original tubes in experienced. Thus, the study of phagocytosis was made using blood samples with different mixture for the study of phagocytosis by two capillary with an inner diameter of 0.8 mm and further processing said mixture, preparation of smears and identification of cellular factors necessary for the subsequent calculation of indicators of phagocytosis, conducted in accordance with the method of the claimed method.

For each of the experimental tubes was determined by PHI (PHI and PI) by the formula (1) and FC (FC and FC) by the formula (2) (result indicators for comparative analysis), comparing them with normal values are given in [4], and have established the presence or absence of change of each of the studied parameters of phagocytosis compared to the norm.

In addition, it was assessed the viability of neutrophils by exclusion Trypanosoma blue according to the method described in [5, S. 50]. With 10 ál of leukocyte suspensions obtained after separation of each of the capillaries 30 - and 120-min period of incubation (for each sample) was mixed with 10 μl of 0.2% aqueous solution Trypanosoma blue on a slide, covered with cover glass and placed under the microscope. Counted the number of blue cells (dead cells) and unstained cells (living cells) in percent of the total number of regarded cells (considered at least 100 cells). For each of the experimental tubes (blood samples) was calculated the average sniego indicator for comparative analysis used the percentage (mean) of dead neutrophils from the total number of regarded cells.

Statistical processing of results was performed using a criterion of Wilcoxon-Mann-Whitney [6].

The research results are summarized in table.1.

From the data table.1 shows that in samples of blood, in which the time between blood sampling from the surveyed child and making the object of phagocytosis in blood with anticoagulant varied from 5 to 30 min, the values of the examined parameters of phagocytosis (FI, FI; FC, FC) was within the range of normal values with the minimum percentage of dead neutrophils (1,1-2,3%). This was not observed statistically significant differences between the values of the indicators examined (phagocytosis indices, the percentage of dead neutrophils) blood samples with retention periods of 5, 15 and 30 min (aggregate data using the criterion of Wilcoxon-Mann-Whitney). When used in the course of preparation of the mixture for the study of phagocytosis of blood samples with anticoagulant, stored more than 30 minutes, there was a statistically significant decrease compared with the norm of all analyzed parameters of phagocytosis, as well as a statistically significant increase in the percentage of dead neutrophils (6.1 to 7.5 percent). It was found that the differences between meant is his and subsequent retention, be statistically significant, since indicators of blood samples 30-45 minute periods of storage.

The selection and justification of the modes and conditions of execution of the action at the stage of processing of the mixture for the study of phagocytosis, as well as actions "selection of cells" was carried out as follows.

Comparative studies of the FAN defined in various modes and conditions of the setting reaction of phagocytosis, had the following children:

- in children with confirmed the clinical-laboratory examination impaired phagocytosis in age from 3 to 12 years (8 persons);

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

The distribution of children in the groups were as follows:

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

group 2 - patients with chronic Wegener (2 people);

group 3 - patients with the syndrome of Jobe (2 people);

group 4 - healthy children (20 people).

According to [7] syndrome Chediak-Higashi refers to congenital immunodeficiencies, accompanied by defects of phagocytosis, which is characterized by a defect in the granules of neutrophils, a significant syndrome Chediak-Higashi (which was the 1st group) were detected during the examination in the hospital, the Children's city hospital No. 1 St. Petersburg /next - Children's hospital No.1/ about recurrent severe infections (all patients were observed recurrent pneumonia, otitis). The diagnosis was confirmed by typical clinical symptom complex: albinism, nystagmus, neutropenia, giant granules in leukocytes, recurrent severe infections. In the study of chemotaxis of peripheral blood leukocytes according to methodology described in [2, S. 333-338], all patients revealed a sharp decrease compared to the norm. In the study of blood using a test of nitrosonium tetrazolium, as described in [3, S. 87-88], all patients experienced a dramatic reduction (compared to normal) recovery narasinga of tetrazole, testified to the deep defect bactericides neutrophils.

Chronic Wegener according to [7] refers to congenital X-linked to immunodeficiency, characterized by reduced bactericides of neutrophils and a decrease in the release of their active oxygen radicals.

Patients with chronic Wegener (2 boys, who was 2nd group) were detected during the examination in the hospital children's hospital No.1 regarding recurrent abscesses, soft tissue and internal organs (one service in the study of blood using a test of nitrosonium tetrazolium [3] found a sharp decline in bactericides of neutrophils compared to the norm. In the study of chemotaxis by the method of [2] abnormalities none of the patients were not identified. In addition, the diagnosis of chronic Wegener from the deceased patient was confirmed by autopsy.

Syndrome Jobe, according to [7], refers to congenital immunodeficiencies, accompanied by defects of phagocytosis. For the vast majority of patients are characterized by reduced chemotaxis of neutrophils with normal rates of bactericides of neutrophils and release their active oxygen radicals.

Patients with the syndrome of Jobe (which was the 3rd group) were detected during the examination in the hospital children's hospital No.1 about the "cold" abscesses subcutaneous tissue, recurrent lymphadenitis, adenilson, not accompanied by redness of the skin and increased skin temperature; recurrent sinopulmonary infections, atypical (staphylococcal) eczema. In the study of chemotaxis of human peripheral blood by the method of [2] in both patients revealed a sharp decline. Variance test of nitrosonium tetrazolium [3] compared with the norm not detected in any of the patients.

The fourth group consisted of 20 children, who are neither in history nor in objectime, instrumental and laboratory methods of variance in health status is not revealed. This group was called "healthy children". Indicators of chemotaxis and test of nitrosonium tetrazolium defined according to the methods of [2; 3], all patients of the 4th group was within the range of normal values.

In the course of determining FAN of the examined children were taken in 10 ml of blood from a vein and was made in a test tube (the original) with a pre-made 5% solution of sodium citrate (2.5 ml per tube). Received the blood with the anticoagulant used for the production of three series of experiments:

I a series of experiments - actions at the stage of preparation of the mixture for the study of phagocytosis, at the stage of processing the above-mentioned mixture (incubation, centrifugation), and the action selection of cells was made using modes and conditions stipulated by the method of the claimed method; as working vessels during cooking and processing the mixture for the study of phagocytosis, followed by separation of cells used capillaries with an inner diameter of 0.8 mm;

II series of experiments - actions at the stage of preparation of the mixture for the study of phagocytosis and on stage obligatorily the inventive method; action "selection of cells" was performed after centrifugation of the mixture for the study of phagocytosis (in accordance with the method of the claimed method) using the techniques provided by known methods (method-prototype [4], a method similar to [1] and so on); as working vessels during cooking and processing the mixture for the study of phagocytosis, followed by separation of cells used centrifuge tubes with a volume of 10 ml;

III a 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 release of white blood cells) and at the stage of processing the above-mentioned mixture (incubation, centrifugation) was made using modes and conditions stipulated by the method of the prototype method; as working vessels during cooking and processing the mixture for the study of phagocytosis used centrifuge tubes with a volume of 10 ml.

For setting the first series of experiments from each source tube (containing the blood of one of the subject child) were selected 0.1 ml in a separate (experienced) tube. In each of the experimental test tubes containing blood with anticoagulant, after 10 min of blood sampling from the surveyed child passing the billion microbial cells in 1 ml (object phagocytosis). Fill the mixture for the study of phagocytosis two capillary with an inner diameter of 0.8 mm (working capacity) and further processing said mixture, preparation of smears and identification of cellular factors necessary for the subsequent calculation of indicators of phagocytosis, conducted in accordance with the method of the claimed method. The results of the timing of the authors of the invention additionally determined the time taken to count on cell smears indicators necessary for the subsequent calculation of indicators of phagocytosis /hereinafter - "the time for counting cell indicators"/ (result indicator for comparative analysis).

For each of the experimental tubes was determined by PHI (PHI and PI) by the formula (1), FC (FC and FC) by the formula (2), CFC by the formula (3) and IBN according to the formula (4) (result indicators for comparative analysis). Comparing the obtained values of the examined parameters of phagocytosis with normal values are given in [4], and comparing judged (as described in the method of the claimed method) about the presence or absence of violations FAN of peripheral blood (disorders of phagocytosis) each of the surveyed children. As result is of lymphocytosis.

While viewing smears under the microscope in the immersion system along with cellular factors necessary for the subsequent calculation of indicators of phagocytosis, it was also calculated the total number of erythrocytes in the reviewed fields of view and to determine the number of erythrocytes per 1 neutrophil (result indicator for comparative analysis).

In addition, for each of the experimental tubes was assessed the viability of neutrophils by exclusion Trypanosoma blue according to the method of [5] are similar to the above during the production of the experiment by choosing the optimal values cause significant temporal parameter. As a result indicator for the comparative analysis used the percentage of dead neutrophils from the total number of regarded cells.

To stage II of a series of experiments from each source tube (containing blood one child) were selected 0.1 ml in a separate (experienced) tube. In each of the experimental test tubes containing blood with anticoagulant, after 10 min of blood sampling in subjects contributed 0.05 ml suspension of staphylococci (culture is the same as in the first series of experiments) in a concentration of 1 billion microbial cells in 1 ml (object f is mportant tubes of 10 ml (working tube) and incubated with each of the operating tubes at a temperature of 37°With the: first - within 30 minutes, the second for 120 min (2 h). At the end of the incubation period the contents of each of the operating tubes were mixed, then each of the operating tubes were centrifuged for 10 min at 1500 rpm After centrifugation was sucked out Pasteur pipette film of leukocytes from the surface sediment of red blood cells, were transferred to skim a glass slide and prepare a smear. Preparation of smears and identification of cellular factors necessary for the subsequent calculation of indicators of phagocytosis, conducted in accordance with the method of the claimed method.

Result indicators for comparative analysis: phagocytosis indices (PI, PC, KFC, IBN); the number of children with diagnosed disorders of phagocytosis; the percentage of dead neutrophils from the total number of regarded cells; the number of erythrocytes per 1 neutrophil; time for calculation of cell parameters were determined as described above for the first series of experiments.

To stage III a series of experiments, the remaining blood in each of the original tubes (containing blood one child) was stirred and centrifuged 10 min at 1000 rpm Plasma with a layer of cells was aspirated with a pipette, placed in a clean centrifuge the tion for 10 min at 1000 rpm After the last centrifugation the supernatant was aspirated liquid, sludge leukocytes resuspendable in medium 199 to a concentration of 10·106cells/ml and added to 0.2 ml of the obtained leukocyte suspensions in two centrifuge tubes with a volume of 10 ml (working tubes). In each of the operating tubes were made of 0.3 ml of a suspension of Staphylococcus (culture is the same as the first series of experiments) in a concentration of 1 billion microbial cells in 1 ml (object phagocytosis), after which was added 0.15 ml of a pool of fresh donor sera AB (IV) group (optionsarray factor) (the time from the moment of blood sampling from the surveyed child before making the object of phagocytosis in leukocyte suspension was made on the results of the timing of the authors of the invention 35-45 min). Mixed components, and incubated with each of the operating tubes at a temperature of 37°With: the first for 30 minutes, the second for 120 min (2 h). At the end of the incubation period in each of the operating tubes was added 5 ml of warmed up to 37°With isotonic sodium chloride solution was shaken and centrifuged for 10 min at 1500 rpm Parallel conducting incubation and centrifugation working tubes (instead of the provided methods of the prototype method serial OS is the licensing conditions of the setting reaction of phagocytosis in all three series of experiments. This modification does not adversely impact on the values of the analyzed result indicators.

Preparation of smears and identification of cellular factors necessary for the subsequent calculation of indicators of phagocytosis, conducted in accordance with the method of the claimed method.

Result indicators for comparative analysis: phagocytosis indices (PI, PC, KFC, IBN); the number of children with diagnosed disorders of phagocytosis; the percentage of dead neutrophils from the total number of regarded cells; the number of erythrocytes per 1 neutrophil; time for calculation of cell parameters were determined as described above for the first series of experiments.

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

The results are shown in table.2, 3.

From the data table.2 shows that the use of the claimed method (first series of experiments) made it possible to detect defects of phagocytosis in all patients with these defects have been previously confirmed by clinical and laboratory examination. At the same time using the proposed method were identified defects of those is the R diseases. Thus, all patients with the syndrome of Chediak-Higashi (1st group) revealed a significant reduction compared to the norm indicators PHI (PHI and PHI), FC (FC and FC) and IBN (differences statistically significant), with a slight (compared to normal) reduced CFC (differences not statistically significant). Chronic Wegener (2nd group) in both patients was observed compared with normal values: significant decline IBN (differences statistically significant); a slight decrease FI (differences not statistically significant) and a more significant reduction FI (differences statistically significant); the reduction KFC (differences statistically significant). In the 3rd group all patients with the syndrome of Jobe results determined using the inventive method showed a significant reduction compared to the norm indicators FI and FI (differences statistically significant), with a slight decrease FC, FC, CFC and IBN (differences not statistically significant). At the same time, the results of the examination, of healthy children with the use of the claimed method showed the absence of violations FAN: all children 4-Oh groups of all analyzed indicators of phagocytosis was in pedacitos:

- in three patients of the 1st group and in one patient of the 3rd group - FI, FI, FC and FC (differences compared with normal values statistically significant);

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

- the patients of the 2nd group and one patient of the 3rd group - KFC (differences statistically significant).

It is not possible to detect defects of phagocytosis in three of the four patients of the 1st group, one of the two patients of the 2nd group and one of two patients of the 3rd group.

In the 4th group (healthy children) have four children were obtained lonoselicesia indicators: FI, FI, FC, FC, CFC, IBN and one child - lonesomejohnnie: FI, FC and IBN (in all cases, differences compared with normal values statistically significant). While none of these children have no diseases and/or various diagnostic, therapeutic and/or preventive procedures, which could cause increase (intercurrent infection, purulent-inflammatory diseases, trauma, surgery, chronic foci of infection, preventive vaccination, etc.,) or decrease (congenital or acquired is of Goritsa neither in history (within 6 months prior to determination FAN), not under the observation of the children in the dynamics (within 2 months after the determination of the FAN) is not marked.

In the third series of experiments were obtained lonoselicesia phagocytosis indices:

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

- the patients of the 1st group - FC (differences statistically significant);

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

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

It is not possible to identify deficiencies in the system of phagocytosis in two of the four patients of the 1st group, one of the two patients of the 2nd group and one of two patients of the 3rd group.

In the 4th group, five children were obtained lonoselicesia indicators: FI, FI, FC, FC, CFC, IBN and two children - lonesomejohnnie: FI, FC and IBN (in all cases, differences compared with normal values statistically significant). While none of these children as well as in the second series of experiments, there were no diseases and is of acetosa, neither in history nor in the watchful waiting (observation period, similar to the second series of experiments). The data table.3 shows that the production reaction of phagocytosis using the declared modes and conditions (first series of experiments) provides the minimum percentage loss of neutrophils (2,3-2,5%) compared with the II and III series of experiments. With the purity of the selection of cells judged by quantitative value admixture of red blood cells (defined as the number of cells per 1 neutrophil), and, accordingly, the time required to count on cell smears indicators necessary for the subsequent calculation of indicators of phagocytosis, in the first series of experiments are comparable with those in III a series of experiments, i.e., when the modes and conditions of the setting reaction of phagocytosis provided by the method of the prototype method (differences of values of the above indices is statistically unreliable). Modes and conditions of the setting reaction of phagocytosis in the second series of experiments provide a significantly lower degree of purity of the selection of cells - the admixture of red blood cells exceeds the first series of experiments in 288-302 times. This leads to an increase in the 3.6-4.2 times the time spent on counting on cell smears on the first index of the first series of experiments).

Experimental studies of the inventors have shown that the use during the production reaction of phagocytosis of capillaries with an inner diameter of 0.2 to 0.8 mm is optimal, since it provides the greatest simplicity of technical operation "selection of cells" in the absence of more linesavannah or loosening the results. When using capillaries with an inner diameter larger than 0.8 mm, there were technical difficulties during the operation "selection of cells". In this case, when the separation capillary was spontaneous (and not induced by touching the tip of the capillary to the subject glass) leaking its contents into the environment. This has led to an increase in impurity plasma to the precipitate of cells by 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 results in 2-3 times. In addition, when using capillaries with an internal diameter of 1.0 and 1.2 mm were obtained lonoselicesia phagocytosis indices in 6-10% of cases and lonesomejohnnie indicators in 5-7% of cases. The use of capillaries with internal Diabetologia strokes low quality in 10-15% of cases microscopic analysis of the entire smear was detected less than 200 neutrophils. When setting reaction of phagocytosis using capillaries with an inner diameter of 0.1 mm were obtained lonesomejohnnie phagocytosis indices in 4-7% of cases, and lonoselicesia in 4-5% of cases.

Achievement provided by the invention technical result is the following. It is known that to obtain the most accurate results of immunological reactions (including reactions phagocytosis), it is necessary to create conditions for a maximum in vitro conservation status of the cells, consistent with their status in vivo. In particular, it is known that damage to the cells by centrifugation occurs when contact with the bottom of the tube, while in suspension, the cells are not damaged even when the hard modes centrifugation (several thousand g) [5]. Optimization of the conditions of production of these reactions can be achieved using various technical tools and techniques (used both in isolation and in combination), in particular, due to:

- use during the production of the reactions of working capacities, allowing to minimize (or even eliminate) the contact of cells with rough poverhnosti cell suspension, that reduces mechanical damage to cells;

- the presence in the incubation environment factors (autologous or heterologous) that contribute to maintaining the viability of the cells and their functional properties, and so on [3].

However, the analysis of patent and scientific and medical literature by the authors of the invention is not discovered methods of setting reaction of phagocytosis, where used technical tools and techniques would provide the possibility of separating the blood of the body on the formed elements directly in the reaction of phagocytosis, and would have been identified and proven the role of the mentioned modification techniques as factor optimization of complex action in respect of the conditions which ensure the preservation of in vitro functional properties of neutrophils responsible for phagocytosis, at the level corresponding individually specific to the organism level in vivo.

The procedure for the determination of the FAN so that the separation of leukocytes and erythrocytes occurs directly during the reaction of phagocytosis, made possible by treatment of the mixture for the study of phagocytosis (stages of incubation, centrifugation) is not in the test tube, and the capillary. PH is) capillary stable liquid layer, retaining walls due to surface tension forces (parietal layer of the capillary). In the parietal layer of the capillary occurs, according to the authors of the invention, the separation of the formed elements of blood on technologies that are characteristic for liquid chromatography, as described, for example, in [8]. The glass wall of the capillary performs the functions of a glass chromatographic media used in practice, in particular for separation by liquid chromatography of a mixture of thermally unstable substances [8]. In terms of the claimed method the formed elements of blood under the action of gravitational forces "slide" towards the bottom of the capillary on the parietal layer, which, acting as a "sliding surface", at the same time provides braking of these elements. Given the different adhesive ability of blood cells, the degree of inhibition of these elements and, accordingly, the speed of their movement are significantly different. In addition, given that the parietal layer of the capillary forms a closed circular surface, the motion vector shaped elements, in particular leukocytes, sent down (to the bottom of the capillary), traumatic contact of cells with steroidov and requires the that the process of separation of leukocytes and erythrocytes already at the stage of incubation of the mixture for the study of phagocytosis occurs with a relatively high intensity. This chromatographic effects (arising when carrying out the reaction in a capillary tube), a considerably smaller area of the bottom of the capillary compared to the bottom of the tube (when equal volumes contained in said tank mixtures for the study of phagocytosis), no mixing of the contents of the capillary (as special operations and result in involuntary shaking when handled during analysis) provide already at the stage of incubation, the synchronicity of the processes of separation of the formed elements of blood and the formation of "erythrocyte pillows and their continuity. In this regard, formed at the incubation stage "erythrocytic cushion has high stability, density and height (the experiments of the inventors on the rationale and conditions for the setting reaction of phagocytosis: I series of experiments).

When setting reaction of phagocytosis in vitro with the observance of other conditions and treatments, provided by the method of the claimed method (experiments of the inventors on the justification of the modes and conditions of production re is very little (especially in the initial period of incubation). At this stage there are some signs of the beginning of the process of formation of "erythrocyte cushion. However, due to the absence of the above specific to the claimed process factors (lack of chromatographic effects, a relatively large area of the bottom of the tube, the presence of mixing as a special operation before incubation and after incubation, and the involuntary shaking of the tube when the manipulation of the analysis) the process of forming erythrocyte cushion has a discrete nature, there is no simultaneity of the processes of separation of leukocytes and red blood cells and formation process "erythrocytic cushion. As a result, erythrocyte cushion formed at the incubation stage, which is highly unstable, has a low density, and its height (to the end of the incubation period) 20-30 times less than the figure achieved in the formulation of the reaction of phagocytosis in the capillary.

When setting reaction of phagocytosis in the capillary at the stage of centrifugation, where there is a sharp intensification of the process of separation of the formed elements of blood, "erythrocyte pillow even more compacted, there is no possibility of its s, dense and high erythrocyte pillow", which completely eliminates the possibility of contact of these cells with the bottom of the capillary, and (given the near-wall effects) and with the walls of the capillary. This, in turn, minimizes the likelihood of injury of cells on the bottom and walls of the capillary, thereby preventing destruction of neutrophils, as well as the emergence of various kinds of violations of their functional properties, including properties that are responsible for phagocytosis (i.e., properties that determine the intensity of processes of chemotaxis and adhesion of these cells, the processes of absorption and digestion of microbes). In particular, the lack of contact with the bottom and walls of the capillary eliminates the possibility revisionaries activation of neutrophils, including revisionaries activate their phagocytic function, due to the processes of adhesion to the bottom and the wall of the capillary.

When setting reaction of phagocytosis in vitro using modes and conditions stipulated in the second series of experiments, the process of separating leukocytes and erythrocytes occurs mainly at the stage of centrifugation. It is at this stage more pronounced than at the incubation stage) becomes and the process of forming red blood cell cushion. ness tubes (the area of the bottom, volume), formed in the tube in the centrifuge "erythrocytic cushion has a significantly lower density, lower height and significantly lower stability in comparison with erythrocyte cushion" capillary), resulting in a relatively high probability of its displacement relative to the bottom of the tube. Under these conditions substantially increases the possibility of contact of leukocytes with the bottom and walls of the tubes, which, in turn, increases the likelihood of trauma to the cells, thereby leading to increased destruction of neutrophils to violations of their functional properties, in particular, to changes in their phagocytic activity (suppression or neesiana activation of phagocytosis).

Thus, the setting reaction of phagocytosis in the capillary using the modes and conditions of the claimed process, allowing you to resolve a number of mechanical and physico-chemical factors that have a negative impact on the FAN and their viability, significantly reduces the probability of obtaining linesavannah and loosening results of the reaction, thereby increasing the accuracy of determination.

Centrifugation of the mixture for the study of phagocytosis in the capillary (I Seri what referirovanija in vitro (II series of experiments). The formation in the centrifugation precipitate blood in the capillary in the form of a column, the height of which exceeds that obtained when using tubes 40 to 50 times, the presence of a distinct boundary between leukocytes and erythrocytes, as well as more stable erythrocyte cushion allows the sludge separation of leukocytes from sediment erythrocytes simple fracture capillary. This greatly simplifies the technical operation "selection of cells". With the purity of the selection of leukocytes (judged by quantitative value admixture of red blood cells in smears) in the first series of experiments comparable with the result obtained in the production reaction of phagocytosis using pre-allocated (by the method of the prototype method) leukocytes (III a series of experiments). This, in turn, ensures the comparability of the conditions and duration microscopic evaluation of the results of the reaction of phagocytosis in its formulation by the method of the claimed method and the prototype method (the first and third series of experiments).

Obtained during centrifugation of the mixture for the study of phagocytosis in vitro (II series of experiments) sediment leukocytes by predstaviteli are much less clearly, and "erythrocytic cushion less stable than in the capillary. Under these conditions, the separation of leukocytes from red blood cells by a simple fracture of the tube (similar to the fault of the capillary) is technically impracticable. The sludge separation of leukocytes from sediment erythrocytes by sucking leukocyte layer does not enable selection of cells with a degree of purity which is necessary and sufficient for an optimal viewing experience strokes. A significant admixture of red blood cells in smears significantly complicates the microscopic evaluation of the results of the reaction of phagocytosis and increases its duration 3.6-4.2 times compared to the first series of experiments).

Thus, the setting reaction of phagocytosis in the capillaries allows the separation process of leukocytes and erythrocytes directly during this reaction (at the stage of incubation of the mixture for the study of phagocytosis), followed by completion of his on-stage centrifugation, and the operation "selection of cells" - after completion of the reaction phagocytosis (after centrifugation). In addition, the implementation process of the separation of leukocytes and erythrocytes do not require special additional operations (as opposed to method-Pia and duration microscopic evaluation of the results of the reaction of phagocytosis in implementing the inventive action sequences (in combination with the modes and conditions of their implementation) are comparable with those, gained through the implementation of the action sequences (in combination with the modes and conditions of their execution) of the prototype method. Thus, the claimed procedure provides relative ease of technical execution of the procedure of determining the FAN and reduces its total duration 30-40 minutes compared to the prototype.

Provided by the method of the claimed method using as one of the components of the mixture to study the phagocytosis of whole blood of the subject body, the separation of leukocytes and erythrocytes directly during the reaction of phagocytosis and secretion of leukocytes after completion of the reaction phagocytosis determine the presence in the reaction mixture along with leukocytes also blood plasma and erythrocytes. On the one hand, this allows us to keep contained in the blood plasma autologous factors, according to the authors of the invention, the preservation of the functional properties of neutrophils, including their phagocytic activity, as well as their viability. In these conditions excludes the necessity of making the mixture for the study of phagocytosis opsonizing factor - donor serum, which, in the opinion of the authors savingsthe disorders of phagocytic function of neutrophils, as well as their viability, due to the negative impact of these immunological factors.

On the other hand, the presence of red blood cells in the mixture for the study of phagocytosis since the reaction of phagocytosis, according to the authors of the proposed solution, may also contribute to the preservation of the functional properties of cells and their viability, because of the following. It is known that erythrocytes have a high sorption surface, resulting in on their surface can absorb various substances [9], including biologically active substances, such as chemokine and cytokines, as well as microbes (in particular, Staphylococcus). The inventors suggest that adsorbed on the surface of the erythrocyte biologically active substances (such as granulocyte-macrophage colony-stimulating factor, interleukins: IL-1, IL-2, IL-3, IL-6, IL-10, IL-18, etc.,) may positively affect functional properties of cells (including those responsible for phagocytosis) and their viability as by direct contact of leukocytes and erythrocytes, and result in the release of these bioactive substances in the reaction medium. In addition, according to the authors of the invention, with the key, in the solution.

Thus, the sequence of steps of the claimed method in combination with the qualitative composition of the mixture for the study of phagocytosis eliminates immunological factors that may affect the phagocytic function of neutrophils and their viability, thus promoting the preservation of the functional properties of neutrophils, including those responsible for phagocytosis. This also contributes to reducing the likelihood of linesavannah and loosening results of the reaction and, therefore, increases the accuracy of determining the FAN.

Preparation of a mixture for the study of phagocytosis taking into account the optimal shelf-life blood of the body (with anticoagulant) before making it the object of phagocytosis contributes to maintaining the viability of neutrophils and their functional properties, including those responsible for phagocytosis, at the appropriate level or close to level, specific to the organism in vivo. When storing blood of the body more than 30 minutes has been a significant violation of the functional properties of neutrophils, which, according to the authors of the invention, due to the death of these cells, violation of the usage during the setting reaction of phagocytosis of blood samples examined body, storage time (before you object phagocytosis) does not exceed the upper limit of the interval of the optimal values of the specified parameter allows you to exclude the possibility of disorders of phagocytic function of neutrophils, as well as reducing their viability, due to the negative effect of this biological factor that significantly reduces the probability of obtaining linesavannah and loosening the results.

In addition, the stated conditions and modes of action provide additional strengthening of the technical result and achieve additional benefits in cases of implementation of the invention. Use during the setting reaction of phagocytosis capillary, inner diameter which is within the optimal interval of values (0.2-0.8 mm), determines the optimal parameters of the lumen of the capillary. Under these conditions, the thickness of the parietal layer of the capillary sufficient to provide a "slip" of leukocytes in the direction to the bottom of the capillary with the exception of the "penetration" parietal layer of these cells (the"punching" parietal layer occurs when using capillaries with internal Diageo additionally reduces the probability revisionaries activation of neutrophils due to their adhesion to the walls of the capillary, as well as the trauma of the specified cells of the capillary wall. The optimal parameters of the lumen of the capillary can also get a precipitate of white blood cells, the volume of which is sufficient for the preparation of high-quality smears containing necessary for counting the number of neutrophils (not less than 200 cells), which further contributes to the creation of optimal conditions for the microscopic evaluation of the results of the reaction. All this further reduces the probability of obtaining linesavannah and loosening results determine the FAN. In addition, the use of capillaries with optimal internal diameter ensures that the difficulties of a technical nature when performing operation "selection of cells" (excludes spontaneous leakage of the contents of the capillary into the external environment) that allows to obtain a leukocyte suspension optimum density (when applied to a glass slide). It additionally helps to reduce the duration microscopic evaluation of results.

Thus, it is stated procedure in combination with the modes and conditions of their implementation eliminates mechanical, physical, chemical, biologypages blood of the body, on the processes of chemotaxis and adhesion of these cells, as well as on the processes of absorption and digestion of microbes. This creates conditions for in vitro conservation of functional properties of neutrophils each specific organism responsible for phagocytosis, at a level that meets or approaching individually specific to the organism level in vivo. This, in turn, allows you to virtually eliminate the possibility of obtaining linesavannah and loosening results of the reaction, thereby increasing the accuracy of the FAN. This prior art is not detected, according to the applicant, the effect prescribed by the invention transformations, characterized by distinctive features of the prototype substantial evidence on the achievement of the technical result.

The method is as follows. Produce blood with anticoagulant in the subject body. In a sterile tube with a pre-made 5% solution of sodium citrate make the blood taken from a finger or from a vein of the subject body with a ratio of a solution of sodium citrate and blood 1:4. Carry out the preparation of a mixture for the study of phagocytosis and processing. If this is bacterial suspension, for example, a suspension of Staphylococcus (which use, for example, daily agar culture of Staphylococcus epidermidis strain 9198 according to the standard) at a concentration of 1 billion microbial cells in 1 ml (object phagocytosis) at a ratio of blood to anticoagulant and an object of phagocytosis 2:1. Fill the mixture for the study of phagocytosis two capillary, inner diameter which is 0.2-0.8 mm Sealed one end of each of the capillaries with a mixture of wax with paraffin and placed both in capillary thermostat in a vertical position. Incubated at a temperature of 37°With each of the capillaries containing the mixture for the study of phagocytosis, one of capillaries within 30 min and the other for 120 min (2 h). At the end of the incubation period, each of the capillaries centrifuged for 10 min at 1500 rpm After centrifugation sever each of the capillaries, for example, by nadpisywania a nail opening the ampoules on the border of the host sediment leukocytes and sediment red blood cells with subsequent breaking of each of the capillaries. Remove the precipitate of leukocytes from the corresponding end portion of each of the disconnected capillaries, for example, by moving the sediment of cells from each capillary naked glass).

Prepare smears of the sediment leukocytes extracted from each of the disconnected capillaries. Prepared smears air-dried, then fixed for 10 min in absolute methyl alcohol and paint Romanovsky-Giemsa Azur-eosin. After staining smears are viewing under the microscope in the immersion system: consider not less than 200 cells (total number of neutrophils). Define each of the strokes number of neutrophils, which came in phagocytosis, and the total number of absorbed Klebsiella microbes /next - cell parameters for subsequent calculation of indicators of phagocytosis"/. Calculate indicators of phagocytosis, which use the following:

1. PHI is defined by the formula (1).

2. FC defined by the formula (2).

Both indicators (PHI and FC) rely on smears prepared from precipitation 30 - and 120-minute incubation (respectively, FI and FI; PC and FC).

3. KFC defined by the formula (3).

4. IBN defined by the formula (4).

The indicators included in the formula (4), - the number of people killed inside Klebsiella microbes (Hy) and the total number of absorbed Klebsiella microbes (HpHp) estimate, for example, by the Oh in [2, S. 384-386] /further - "bacteriological indicators necessary for the subsequent calculation of IBN"/. While Hydetermined by the formula:

where Hpthe number of colony forming units /CFU/ Staphylococcus planting lysed sediment cells, incubated for 30 min;

HP- the number of CFU of Staphylococcus planting lysed sediment cells, incubated for 120 min;

Hy- the difference between the number of CFU of Staphylococcus planting lysed sediment cells, incubated for 30 min, and the number of CFU of Staphylococcus planting lysed sediment cells, incubated for 120 minutes

Compare the obtained values of phagocytosis with normal (for the type of organisms, which include the target organism) values. For examination of patients and healthy people use, for example, normal values given in [4]. Establish the presence or absence of change of each of the studied parameters of phagocytosis compared to the norm. When the values of all the studied parameters of phagocytosis in normal limits make the conclusion about the absence of violations of the FA is for make a conclusion about the existence of irregularities FAN of this organism.

The invention is illustrated by the following examples.

Example 1

Patient Serge So, 7 years. Addressed in the outpatient Department No. 41 children's hospital No.1 with complaints of cough, fever up to 38.5°C. Ill for 5 days. Received symptomatic therapy (fever and expectorants), but the condition worsened, and therefore appealed to children's hospital No.1. In the anamnesis the patient noted recurrent pneumonia, furunculus, adenomegaly, osteomyelitis from an early age (age 1 month). The child also seen a neurologist about lag in psychomotor development, nystagmus.

The patient carried out a determination of the FAN using the inventive method and the prototype method. In the course of determining FAN of the examined child took 10 ml of blood from a vein and introduced into a sterile tube (source) with pre-filled in the amount of 2.5 ml of 5% solution of sodium citrate. Received the blood with the anticoagulant used for the production reaction of phagocytosis by the method of the claimed method and by the method of the prototype method.

During the determination of the FAN using the claimed method from the original tubes containing blood with anticoagulant, took 0.1 m is a (time determined by the timing results of the inventors) added 0.05 ml of a suspension of Staphylococcus (daily agar culture of Staphylococcus epidermidis, pieces 9198 according to the standard) at a concentration of 1 billion microbial cells in 1 ml Filled mixture for the study of phagocytosis two capillary with an inner diameter of 0.8 mm were Soldered one end of each of the capillaries with a mixture of wax with paraffin and placed both in capillary thermostat in a vertical position. Incubated at a temperature of 37°With each of the capillaries containing the mixture for the study of phagocytosis: one of the capillaries within 30 min and the other for 120 min (2 h). At the end of the incubation period, each of the capillaries were centrifuged for 10 min at 1500 rpm After centrifugation separated each of the capillaries by nadpisywania a nail opening the ampoules on the border of the host sediment leukocytes and sediment red blood cells with subsequent breaking of each of the capillaries. Extracted residue of leukocytes from the corresponding end portion of each of the disconnected capillary by moving the sediment of cells from each capillary to skim a glass slide (touching the tip end part of the capillary containing the leukocytes, slides).

Prepared smears of the sediment leukocytes extracted from each of the disconnected capillaries. Prigotovleniya Azur-eosin. After each staining of smears were viewed under the microscope in the immersion system: counted 200 cells (total number of neutrophils). Defined on each of the strokes cellular factors necessary for the subsequent calculation of phagocytosis indices:

Calculated parameters of phagocytosis: PHI by the formula (1), FC - by the formula (2), KFC - by the formula (3):

Identified bacteriological indicators necessary for the subsequent calculation of IBN, by planting dense nutrient medium (agar) washed and lysed sediment cells, incubated for 30 min and within 120 min (method [2]):

Calculate the number of people killed inside Klebsiella microbes (Hy) as the difference between the number of CFU of Staphylococcus planting lysed sediment cells, incubated for 30 min, and the number of CFU of Staphylococcus planting lysed sediment cells, incubated for 120 min, according to the formula (5):

Hy=156-101=55 CFU Staphylococcus

Computed a measure of IBN according to the formula (4):

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

In parallel, the patient was estimation of the FAN according to the method of the prototype method [4]. While remaining in the initial tube blood with anticoagulant mixed, after which it was centrifuged 10 min at 1000 rpm Plasmas with a layer of cells was aspirated with a pipette, placed in a clean centrifuge tube and added 10 ml of medium 199. Double-washed cells in medium 199 by double centrifugation for 10 min at 1000 rpm After the last centrifugation was aspirated by pipette the supernatant liquid and the portion of the cell suspension, leaving the centrifuge tube 0.2 ml of cell suspension in medium 199. Then in a test tube, with the Academy of Sciences of the claimed method) at a concentration of 1 billion microbial cells in 1 ml, then added 0.15 ml of a pool of fresh donor sera AB (IV) group (the time from the moment of blood sampling from the surveyed child before making the object of phagocytosis in leukocyte suspension was, according to the timing of authors of inventions, 43 min). Mixed components and thermostatically the mixture for the study of phagocytosis 30 min at a temperature of 37°C. Upon expiration of the time specified in a test tube was added 5 ml of warmed up to 37°With isotonic sodium chloride solution was shaken and centrifuged 10 min at 1500 rpm after centrifugation the supernatant was removed and made the strokes of the leukocyte suspension, the remnants of which was placed in a thermostat at 37°to continue incubation for 90 min (total incubation time 120 min). Then smears prepared again from the precipitate 120-minute incubation. Smears from the precipitation of leukocytes (after 30-minute and 120-minute incubation) were prepared low fat slides. Fixation and staining of the slides produced as well as the definition of a FAN of the claimed method. On each of the strokes identified cellular factors necessary for the subsequent calculation of indicators of phagocytosis, and calculated indicators Pahoa subsequent calculation IBN, and calculated IBN similar to the claimed method.

Results definitions and calculations are presented in the summary table.4.

Comparison of results of determination of normal values [4] showed that the obtained values of all studied phagocytosis indices were within the range of values of corresponding normal values. Thus, the method-prototype revealed no violations of the FAN. The duration of the procedure definition, the result of the timing of the authors of the invention, amounted to 198 min (3.2 h).

A comprehensive examination of the patient. At clinical examination noted albinism, silvery glow hair when illuminated by an electric lamp, no deposition of pigment in the retina. On the chest x-ray revealed infiltration in the lower lobe of the right lung.

The results of laboratory tests

The blood analysis revealed neutropenia:

b 116 g/l; Ág 3,8·1012/l; leukocytes 5,6·109%; platelets 180·1012/l; stab 7%; segmented 8%, eosinophils 4%; lymphocytes 70%; basophils 2%; monocytes 9%; ESR 25 mm/h

In the immunological identified:

1) a slight decrease relative activities);

CD4 35% (normal 31-46%);

CD8 28% (normal 26-40%);

CD16 15% (normal 9-16%);

CD20 18% (normal 11-16%);

CD25 25% (normal 13-24%);

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

Ig G 690 mg % (normal 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 (norm 100-250 µg/ml);

C3 850 µg/ml (norm 700-1800 mg/ml);

Sa of 0.07 µg/ml (normal range 0.05 to 0.15 µg/ml);

C4 400 µg/ml (norm 200-500 µg/ml);

Sa of 0.025 ág/ml (normal range 0,01-0,03 µg/ml);

4) spontaneous and induced cytokine production were slightly different 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 of chemotaxis of neutrophils:

index migration under the influence of FMLP 0.2 (norm 2,6-2,8);

7) slight decrease test of nitrosonium tetrazolium:

granules formazan identified in 8% neutrophils (normal 11-40%).

Taking into account medical history, clinical picture and conducted a comprehensive laboratory obsm [7] refers to congenital immunodeficiencies, accompanied by defects of phagocytosis, the results of a comprehensive survey confirmed the results of determining the FAN obtained by the claimed process, and helped to interpret the results of determining the FAN on the method prototype as lonoselicesia.

Example 2

Patient Tatiana S., age 14. Addressed in the outpatient Department No. 41 children's hospital No.1 with complaints pustular rash on the skin, increased body temperature to 37.8°C. the Deterioration noted two weeks ago: pustular rash on the skin (boils) has appeared on various parts of the body (right shoulder, abdominal wall, thighs). She received treatment at the district hospital (headbands with furatsilina, various ointments, lancing boils), but boils appeared on new areas of the body. In the anamnesis of a patient with 1,5 years there has been a recurrent furunculosis with exacerbations in fall and winter.

The patient carried out a determination of the FAN using the inventive method and the prototype method similar to example 1.

When determining the FAN using the claimed method were used capillaries with an inner diameter of 0.6 mm, the Time from the moment of blood sampling from the surveyed child up in the accounts given in the summary table.5.

When comparing the obtained values of phagocytosis indices with normal values revealed a significant reduction FC, FC and IBN with a slight decrease FI, FI (CFC slightly increased), testified to the presence of a defect in the phagocytic immunity, namely disorders as initial and final stages of the process of phagocytosis. The determination spent 152 minutes (2.5 hours).

In parallel, the patient carried out a determination of the FAN using the prototype method. The time from the moment of blood sampling from the surveyed child before making the object of phagocytosis in leukocyte suspension was 45 minutes

Results definitions and calculations are presented in the summary table.5.

The matching results obtained using the prototype method parameters phagocytosis of normal values were similar to the corresponding data comparison results obtained by the claimed process (significant reduction FC, FC and IBN, a slight decrease FI, FI, increasing CFC). It is possible to make the similar conclusions of the claimed method (the presence of a defect in the phagocytic immunity; violations as the>is roweena comprehensive examination of the patient analogously to example 1. At clinical examination revealed the presence of furuncles on the right hip, right leg, left arm; the body temperature is 37°C. Physical development of the child is age appropriate. Re-investigation of blood sugar and diabetes curves revealed no abnormalities.

The results of laboratory tests

The blood analysis revealed neutrophilic leukocytosis with a left shift: b 135 g/l; Er 4,1·1012/l; leukocytes 15,7·109/l; platelets 220·1012/l; young 3%; stab 15%; segmented 48%, eosinophils 2%, lymphocytes 25%, monocytes 7%; ESR 32 mm/h

In the immunological identified:

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

CD3 56%;

CD4 34%;

CD8 27%;

CD16 12%;

CD20 19%;

CD25 25%;

2) concentration of antibodies corresponded to the age norm:

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

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

Ig A 230 mg % (normal 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;

Sa 0.12 µg/ml;

C4 is but 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:

index migration under the influence of FMLP 2,6;

7) reduction test of nitrosonium tetrazolium:

granules formazan detected in 6% of neutrophils.

Comprehensive survey allowed us to conclude that a cause of recurrent boils in a child is the syndrome - defect in phagocytosis, since when assessing other elements of the immune system abnormality is not detected. Thus, in this case, was the coincidence of the results of determination of the FAN using the inventive method and the prototype method. The results of the phagocytic reaction correlated with data from complex surveys.

Example 3

Child of Nicholas P., age 16. The patient was examined in the outpatient clinic No. 41 children's hospital No.1 in the order of examination. Complaints during the inspection did not show. History rare (1-2 times per year) SARS.

The boy had spent the definition of FAN using the inventive method and the prototype method similar to example 1.

During the determination of the FAN according to the method of the claimed method were used KTA phagocytosis in blood with anticoagulant was 5 minutes

Results definitions and calculations are presented in the summary table.6.

When comparing the obtained values of phagocytosis indices with normal values of deviations from the norm have been identified that have led to the conclusion about the absence of disturbances in the system of phagocytosis in the examined children. The duration of the procedure definition - 136 min (2,3 h).

In parallel, the boy carried out a determination of the FAN according to the method prototype. The time from the moment of taking blood from the subject of the child before making the object of phagocytosis in leukocyte suspension was 36 minutes

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

The result of the comparison obtained by using the prototype method of data with normal values revealed a significant reduction FC, FC and IBN, on what basis it was possible to conclude that the child has a serious breach in the system of phagocytosis (at the initial and final stages of the process of phagocytosis). The duration of the procedure definition 178 min (3.0 hours).

A comprehensive examination of the child. At clinical examination revealed a flat foot. Deviations from other organs and systems not found.

b 145 g/l; Er 4,5·1012/l; leukocytes 6,3·109%; platelets 250·1012/l; stab 3%; segmented 53%, eosinophils 2%; lymphocytes 37%, monocytes 5%; erythrocyte sedimentation rate of 3 mm/h In the immunological:

1) the content of T - and b-lymphocytes consistent with the norm:

CD3 40%;

CD4 43%;

CD8 32%;

CD16 15%;

CD20 12%;

CD25 21%;

2) concentration of antibodies corresponded to the age norm:

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

Ig M 112 mg % (normal 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;

Sa of 0.11 μg/ml;

C4 410 µg/ml;

Sa of 0.015 µg/ml;

4) spontaneous and induced cytokine production 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:

index migration under the influence of FMLP 2,7;

7) test nicrosini tetrazolium corresponded to the norm:

granules formazan identified in 38% of neutrophils.

Comprehensive examination showed the following. The REB is aniah in the immune system, not marked. In particular, no known diseases that could lead to a decrease in phagocytosis. No therapeutic, diagnostic and/or prophylactic procedures, which could cause a decrease in phagocytosis. Thus, the results of a comprehensive survey that indicated no violations of the FAN, confirmed the determination results obtained by the claimed process, and given the opportunity to consider the results of determining the FAN on the method prototype as lonesomejohnnie.

The claimed method was used in the survey of 342 children aged 3 to 15 years, which was held at the children's hospital No.1 (in the outpatient clinic No. 41). For comparison, the definition of FUN was performed using the prototype method [4].

All children were also given a comprehensive examination using a variety of clinical, instrumental and laboratory methods, including immunological methods (determination of the number of T - and b-lymphocytes; the concentration of immunoglobulins a, M, G; content of components of the complement system; determination of spontaneous and induced cytokine production; determination of the level of the CEC; the definition of a FAN is in the peripheral blood by the method, given in [2], and by setting the test with nitrosonium tetrazolium according to the method described in work [3]. Taking into account medical history, clinical picture and conducted comprehensive surveys were delivered to clinical diagnoses (hereinafter - "the results of the control of complex examination /KCO/"). While the surveyed children were divided into the following groups:

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

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

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

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

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

VI group - patients with congenital heart defects (48 persons);

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

VIII group - practically healthy children (15 people).

Statistical processing of results the survey was conducted using criteria Fisher's exact and Wilcoxon-Mann-Whitney [6].

Received financial p the mi diseases (according to groups) and in healthy children using the inventive method and the prototype method. In table. 8 shows the summary data characterizing the effectiveness of the inventive method and the prototype method.

From table.7 shows that violations of the FAN have been confirmed by KCO in 22 children (2 patients with the syndrome of Chediak-Higashi (group I); and 12 patients with recurrent furunculosis (group II); 5 patients with recurrent lymphadenitis (group III); in 3 patients with recurrent otitis, sinusitis, pneumonia (group IV)).

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

Using the prototype method identified deficiencies in the system of phagocytosis (confirmed KCO) in 15 patients (1 patient with syndrome Chediak-Higashi (I gr.); 9 patients with recurrent furunculosis (II gr.); in 3 patients with recurrent lymphadenitis (III gr.) and in 2 patients with recurrent otitis, sinusitis, pneumonia (IV g)). 7 patients with confirmed KCO violations FAN (1 Bologoe the lymphadenitis (III gr.) and in 1 patient with recurrent otitis media (IV gr.)) values of phagocytosis, obtained when using the prototype method, consistent with normal values (lonoselicesia the results of determination). In addition, identified using the prototype method violations FAN in 43 cases were not confirmed by the results KCO (in 15 patients with chronic gastritis, duodenitis, biliary dyskinesia (V g): in 11 cases lonoselicesia the determination results, in 4 cases - lonesomejohnnie; in 3 patients with congenital heart defects (VI gr.): in 2 cases lonoselicesia results, in one case lonesomejohnnie; in 21 patients with disorders of the musculoskeletal system (group VII): in 16 cases lonoselicesia results in 5 cases lonesomejohnnie and have 4 healthy children (VIII gr.): in 3 cases lonoselicesia results, in one case lonesomejohnnie).

As follows from the data table.8, the claimed method provides for its implementation 100% accuracy for the determination of the FAN, which is 14.6% more than the accuracy using the prototype method. When it is confirmed KCO violations FAN the determination results by the method prototype were regarded as erroneous in 31.8% of cases, and in the absence of disturbances in the system of phagocytosis (podtverzhdennost procedures for determining the FAN is on average 1.3 times in comparison with the method of the prototype (by eliminating the need for prior separation of leukocytes and erythrocytes, and selection of cells as special operations during the mixture preparation for the study of phagocytosis, the implementation of the separation of leukocytes and erythrocytes directly during the reaction of phagocytosis and simplify operations "selection of cells"). However, the duration of microscopic evaluation results comparable with results of the prototype method. Thus, the claimed method in its implementation provides a highly accurate determination of the FAN when the relative ease of technical execution and a relatively small duration analysis.

Sources of information

1. Chernushenko E. F., Kolosova HP Immunology and immunopathology of pulmonary diseases. - Kiev: Health, 1981. - S. 169.

2. Immunological methods: TRANS. with it. Ed., Primes. - M.: Medicine, 1987. - S. 333-338, 384-386.

3. Douglas S. D., kui P., the Study of phagocytosis in clinical practice: TRANS. from English. - M.: Medicine, 1983. - S. 63-64, 87-88.

4. Laboratory methods in the clinic: a Handbook /in. A. Menshikov, L. N. Delektorskaya, R. P. Zolotnitskaya and others, Ed. by C. C. Menshikov. - M.: Medicine, 1987. - S. 310-311 (prototype).

5. Lymphocytes: Methods: TRANS. from English. Ed. j.Claus. - M.: Mir, 1990. - S. 29.50.

6. Gubler E. C. Computational methods of analysis and recognition of pathological process. W. B. Saunders Company, 1996. - P. 561-609.

8. Lurie A. A. Chromatographic materials: a Handbook. - M.: Chemistry, 1978. - S. 190-191.

9. Hematology childhood: a Guide for physicians. /Ed. by N. A. Alekseeva. - SPb: Hippocrates, 1998. - S. 97-105.

1. The method of determination of phagocytic activity of neutrophils peripheral blood, including the blood with the anticoagulant, the preparation of a mixture for the study of phagocytosis containing leukocytes and the object of phagocytosis, secretion of cells, processing the mixture for the study of phagocytosis incubation and centrifugation, preparation of smears from obtained after centrifugation precipitation with subsequent determination on each of the strokes in the number of neutrophils, which came in pazitos, and the total number of absorbed Klebsiella microbes, the calculation of rates of phagocytosis and the comparison of obtained values of phagocytosis with normal values, characterized in that during the mixture preparation for the study of phagocytosis first make the object of phagocytosis in blood with anticoagulant no later than 30 minutes after its intake, then fill obtained from the 37°With each of the capillaries, one of capillaries incubated for 30 min and the other for 2 h, and centrifugation of each of the capillary for 10 min at 1500 rpm, the selection of leukocytes produce directly after centrifugation by the end of each of the capillaries on the border of the host sediment leukocytes and sediment erythrocytes and sediment extraction of cells from the corresponding part of each of the disconnected capillaries, preparation of smears carried out precipitation of leukocytes and phagocytosis indices used phagocytic index, phagocytic number, the coefficient of phagocytic number and index bactericides neutrophils.

2. The method according to p. 1, characterized in that during the preparation of the mixture for the study of phagocytosis and processing use capillaries with an inner diameter of 0.2 to 0.8 mm



 

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2 ex

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EFFECT: improved assay method.

3 tbl, 3 ex

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3 ex

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EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

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EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

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2 ex

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EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

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EFFECT: high accuracy of diagnosis.

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