Method for determination of functional activity of phagocytes (faf)

FIELD: medicine, namely immunology, allergology, dermatology, microbiology; veterinary.

SUBSTANCE: claimed method includes providing of control mixture (CM); blood sampling with anticoagulant; providing of mixture for phagocytosis investigation (PhIM) by introducing in tested blood sample preliminary prepared 0.1 % solution of chromogenic reagent, namely tetrasolium nitroblue (NTB) dissolved in 0.9 % sodium chloride solution; PhIM incubation; preparation of smears from incubated followed by determination of amount of formasan-positive phagocytes in percents based on total phagocyte amount and FAF evaluation by comparison obtained values with normal ones. To produce CM part of preliminary prepared NTB solution with volume of at least 0.05 ml is blended with yeast Saccharomyces cerevisiae and obtained CM is held for at least 6 min at temperature of (20±2)°C. Then CM color is analyzed and rest part of NTB solution is used for PhIM preparation only in presence of pink color in CM. Method of present invention makes it possible to increase accuracy of FAF determination by 25-33 %.

EFFECT: method for FAF determination with improved accuracy.

7 cl, 8 tbl

 

The invention relates to medicine, namely to immunology, Allergology, pulmonology, dermatology, Microbiology, veterinary medicine, and can be used to assess the suitability of a chromogenic reagent - microcinema of tetrazole /hereinafter - the "PCT"/ for use in the reaction of phagocytosis in assessing the immune status of healthy and diseased organism.

Known methods of determining the functional activity of phagocytes (hereinafter "fat"/ test results with the recovery of HCT - so-called NBT-test [1, 2].

So, there is a method of determining fat children in normal and purulent bacterial infections [1]. The formulation of the NBT-test according to the method similar [1] is as follows. Prepare the mixture for the study of phagocytosis /"CIF"/. To 0.1 ml of heparinized blood taken from a finger of the subject person) add 0.1 ml of 0.075% solution of HCT. Incubated Seth for 10 min at 37 ° °C. After incubation, record the indicated mixture of 50% formalin solution, diluted in 0.9% sodium chloride solution for 3 minutes, Centrifuged Seth for 5 min at 750 rpm the Supernatant liquid removed from the sludge prepare smears. Fix smears in alcohol-formalin for 30 s Domracheva kernel 0.1% solution of neutral red dissolved in 0.5% chloride solution three is. Under the microscope using the oil immersion system totals 200 phagocytes and determine the percentage of positively reacting with CNT cells, in the cytoplasm which marked the formation of granules formazan (purple-blue) /next - formazan-positive cells"/. Fat evaluated by comparing the obtained values of the NBT-test with similar indicators for healthy children, which, according to the authors of the method-analogue of [1], average (14.4V±0,68)%, varying within 4-33%. As a result of this mapping by the authors consider how similar it was found, in particular, that in infectious diseases has significantly increased values of HCT test.

Closest to the claimed solution of the essential features is the method of determining fat [2]. The method adopted for the prototype, as follows. Prepare Seth. Thus, 0.1 ml of blood mixed with 0.1 ml of 0.1% solution of HCT in 0.9% solution of sodium chloride (0.15 M NaCl). Incubated Seth for 20 min at 37°; after incubation this mixture is thoroughly mixed. From the contents of the tubes obtained after incubation Seth, prepare smears and produce microscopy. Determine in a number of formazan-positive phagocytes in percent of the total number of phagocytes. Frozenat by comparing the obtained values with the norm, which, according to the authors of the prototype method [2]is about 30%.

However, the known method is similar to [1] and the method-prototype [2] does not allow to obtain a technical result achieved when using the inventive method, for the following reasons. The results of the clinical observations of the invention (definition fat during a comprehensive survey of 467 children) showed that in the formulation of NBT-test using known methods [1, 2] in 25-33% of cases have been recorded results that do not correlate with data from a comprehensive survey and regarded as false negative. It was found that in these cases, replacement of the used CNT sample on the sample corresponding to the criterion of suitability (pursuant to the stated method), resulted in the receipt of the results of the NBT-test, fully confirmed by the results of a comprehensive survey. Thus, the authors claimed process came to the conclusion about the correlation between the activity used in the reaction phagocytosis of CNT sample and the results of determination FAF. This assumption is confirmed by the following. It is known that the PCT is a redox indicator that, in doing faguoqitirute cells, undergoes reduction to insoluble formazan under the influence of the oxidase of free nucleotides OVER on; H and NADP·N [1]. It is known that the PCT retains its activity only under certain storage conditions (in the dark, in a sealed package) [3]. There is evidence of poolability CNT [4]. In a number of works relating to technology use reagent PCT, in particular in [5], the recommendations about the need for daily cooking again chromogenic substrates containing CNT. According to the authors of the proposed method, the CNT sample, storage conditions which have been violated (in one way or another), changes the chemical structure of preventing the transformation of the PCT in granules formazan under the influence of the respective enzymes (OVER·H and NADP·N). This, in turn, prevents the accurate assessment of fat using HCT-text. Thus, the absence of a preliminary assessment of the suitability of the sample for PCT applications in the reaction phagocytosis leads to a decrease in the accuracy of determination FAF due to the inability to obtain reliable results of the NBT-test using samples PCT lost the ability to form the restored form.

Object of the invention is to provide a method for determining fat, providing the ability to pre-assess the ability of the PCT to chemical transformation in formazan under the influence of redox enzymes by use the output test cell culture, the structural and functional characteristics of the membranes and superficial layer of cytoplasm which contribute to the manifestation of macroscopically distinguishable color when the positive result of the reduction reaction of CNT in formazan.

The problem is solved in that in the method of determining fat, including blood with anticoagulant, cooking Seth by making the blood sample is pre-prepared with 0.1% solution of HCT, dissolved in 0.9% sodium chloride solution, incubation Seth, preparation of smears from preincubating Seth with subsequent determination of the amount of formazan-positive phagocytes in percent of the total number of phagocytes and evaluation FAF by comparing the obtained values with normal values, according to the invention before cooking Seth cook the control mixture (hereinafter "CS"/. For this part of a pre-prepared solution of NCT of not less than 0.05 ml is mixed with the yeast Saccharomyces cerevisiae and maintain the received CC for at least 6 minutes at a temperature of (20±2)°C. Then analyze the color of the COP and use the rest of the CNT solution for the preparation of Seth only if the COP pink colouring. The most effective to mix with the yeast Saccharomyces cerevisiae part of a pre-prepared solution of CNT volume of 0.05-0.1 ml In the line is the use for the preparation of the COP of the yeast Saccharomyces cerevisiae dry is best to mix them with the CNT solution at a concentration of 50-250 mg yeast/ml NBT, and if you are using the yeast in the form of a suspension concentrate (2,5-12,5)×108yeast cells/ml of HCT /hereinafter - "the working concentration CS/. The most effective is keeping the received CC for 6-10 minutes are also effectively additionally centrifuged Seth, for example for 5 min at 750 rpm, and prepare smears from obtained after zentrifugenbau sediment.

To select and justify the optimal values cause significant parameters of the proposed method, which achieves the highest efficiency of the proposed measure PCT for use in the setting of HCT-test /hereinafter - "the proposed criterion of suitability"/, were carried out two-stage test. The first stage was assessed by visual analysis of the COP qualitative component of the proposed measure is the presence of pink colouring KS /hereinafter "compliance test"/. In the second stage confirmed the validity of the measure by identifying the correlation between the estimated visually qualitative component of the specified criteria and evaluate clinical and laboratory methods the quantitative component of this criterion (confirmed metric values FAF).

During testing it was delivered four series of experiments in which ASCS were adavani 19 samples nst, obtained from multiple sources, stored under different conditions for different times. In each series of experiments we used samples with similar conditions and shelf life:

I a series of experiments - CNT samples from consignments received before testing; no apparent violations of the packaging (10 samples);

II series of experiments - CNT samples of the same parties as in the first series of experiments that have been tested for compliance with the proposed measure with a positive result stored (after testing for compliance) exposed to light for 7 days (3 samples);

III a series of experiments - CNT samples of the same parties as in the first series of experiments that have been tested for compliance with the proposed measure with a positive result stored (after testing for compliance) in a package with broken seals within 30 days (3 samples);

IV a series of experiments - CNT samples from different batches, which were kept in the laboratory where the trials were carried out over 10 years; without apparent violations of packaging (3 samples).

The tests were carried out in the immunological laboratory of the Children's city hospital No. 1 St. Petersburg /hereinafter - "children's hospital №1"/. Visual analysis of the COP in the first phase of testing was performed using the method of expert evaluation. In tests of the first stage took part 60 lab what's workers aged 18 to 75 years, 36 of them are people with 100% vision; 15 people with myopia (-2)-(-8)D; 9 patients with hyperopia (+2)-(+6) D /"experts"/. Each of the experts were asked to describe visually distinct color COP, prepared according to the claimed method using each of the samples nst (each series). In accordance with the claimed method is believed that the sample CNT corresponds to the proposed measure when determining the color of the COP of this sample CNT as "pink". The color investigated the COP considered adequate, if the assessment results are matched at 100% of the experts is "adequate expert evaluation of color KS"/.

During testing of the first stage using a visual analysis of the COP, prepared according to the claimed method using each of the samples CNT:

- established the fact of presence or absence of pink colouring KS each specific sample PCT /also referred to as, respectively, "positive or negative result of visual analysis/ conditions guaranteed adequate expert evaluation of color COP;

for all CNT samples, for which he received a positive result for visual analysis /hereinafter referred to as "CNT samples corresponding to the proposed criteria of suitability," "positive samples PCT" "PCT"/, who was issledovalas visually perceptible pink KS each of these CNT samples with different values of the declared parameters, and according to the total results of the research were determined intervals the optimal values of these parameters.

Under the guaranteed adequate expertise to understand the color of the COP of each specific positive CNT sample when the concentration of the yeast Saccharomyces cerevisiae 250 mg yeast/ml NBT - when using yeast in dry form or 12.5×108yeast cells/ml NBT - when using yeast as a suspension when the amount of insertion of the CNT solution 0.1 ml and time-keeping COP 10 min at a temperature of 22°C (upper limit values of the intervals of the optimal values of these parameters). The visual appearance of pink color COP positive CNT samples were investigated under conditions of varying concentrations of yeast at constant (optimal) amount of insertion of the CNT solution and constant (optimal) time keeping the COP at a temperature of 20°With; in terms of the variation of the amount of insertion of the CNT solution at a constant (optimal) yeast concentration and constant (optimal) time keeping the COP at a temperature of 20°and in conditions of varying time-keeping COP at a temperature of 20°at constant (optimal) yeast concentration and constant (optimal) amount of proposed solution The PCT. In addition, under conditions of varying temperature and time in which derivare COP at each of the temperatures of the investigated range at constant (optimal) yeast concentration and constant (optimal) amount of insertion of the CNT solution for each positive sample CNT recorded the appearance of pink colouring COP this sample had been examined visually perceptible, and the possibility of adequate expert evaluation of color COP.

For the experiments of the first and second stages of the test was previously prepared 0.1% solution of each of the samples NBT dissolved in 0.9% solution of sodium chloride. At the first stage part of a pre-prepared solution of each of the CNT samples used for the preparation of the COP on the ten options listed below (the rest of the specified solution used to prepare the CIF in the second stage of the test). Each of the variants were prepared based on the method of the claimed method, taking into account the specific quantitative values of constant and variable parameters set by the conditions setting of the experiment in each of the options (hereinafter referred to as the "pilot test"/:

option 1:the yeast Saccharomyces cerevisiae in a dry form; the concentration of CA 250 mg yeast/ml PCT when the amount of insertion of the CNT solution 0.1 ml;
aging time COP 10 min at 22°C;
option 2:the yeast Saccharomyces cerevisiae as a suspension; koncentracija KS 12,5×108yeast cells/ml NBT in the amount of applied p is the target HCT 0.1 ml;
aging time COP 10 min at 22°C;
option 3:the yeast Saccharomyces cerevisiae in a dry form; the variation of the concentrations of CA in the range 5 are 300 mg yeast/ml NBT (i.e. 5 mg of yeast/ml NBT) at constant volume made of CNT solution 0.1 ml;

aging time COP 8 min at 20°C;
option 4:the yeast Saccharomyces cerevisiae in the form of a mist; the variation of the concentrations of CA in range (0,25÷15)×108yeast cells/ml NBT (i.e. 0,25×10 yeast cells/ml NBT) at constant volume made of CNT solution 0.1 ml;
aging time COP 8 min at 20°C;
option 5:the yeast Saccharomyces cerevisiae in a dry form; the variation of the amount of insertion of the CNT solution in the range from 0.01 to÷0.2 ml (i.e 0,025 ml) at a constant concentration of 150 mg yeast yeast/ml of HCT;
aging time COP 8 min at 20°C;
option 6:the yeast Saccharomyces cerevisiae in the form of a mist; the variation of the amount of insertion of the CNT solution in the range from 0.01 to÷0.2 ml (i.e 0,025 ml) at a constant concentration of yeast 7,5×108yeast cells/ml of HCT;

aging time COP 8 min at 20�B0; C;
option 7:the yeast Saccharomyces cerevisiae in a dry form; the variation of the time-keeping of the COP within 1-15 minutes (i.e. 1 min) at 20°at a constant concentration of 150 mg yeast yeast/ml NBT and at constant volume made of CNT solution 0.1 ml;
option 8:the yeast Saccharomyces cerevisiae in the form of a mist; the variation of the time-keeping of the COP within 1÷15 min (i.e. 1 min) at 20°With; with constant yeast concentration of 7.5×108yeast cells/ml NBT and at constant volume made of CNT solution 0.1 ml;
option 9:the yeast Saccharomyces cerevisiae in a dry form; the variation of the temperature, which was maintained to the COP within 2÷37°With (i.e. 2°With; the estimated interval of the optimal value and the upper boundary values of the investigated range - 1° (C)when the variation of the time-keeping of the COP at each of the temperatures of the investigated range within 1÷15 min (i.e. 1 min) at a constant concentration of 150 mg yeast yeast/ml NBT and constant volume made of CNT solution 0.1 ml;
option 10:the yeast Saccharomyces cerevisiae in the form of a mist; the variation of the temperature, which was maintained to the COP within 2÷37°With (what ncremental 2° With; the estimated interval of the optimal value and the upper boundary values of the investigated range - i.e. 1° (C)when the variation of the time-keeping of the COP at each of the temperatures of the investigated range within 1÷15 min (i.e. 1 min) at a constant concentration of yeast 7,5×108yeast cells/ml NBT and at constant volume made of CNT solution of 0.1 ml.

For the purpose of visual analysis (for presentation to the experts) used transparent centrifuge tubes with a volume of 10 ml containing the COP of each of the cooking options for each of the samples PCT /hereinafter - "pilot tube"/. In parallel with each of the experimental samples in similar tubes (hereinafter "control tube"/ prepared control sample. At the same time as the source component for the preparation of the test sample used the yeast Saccharomyces cerevisiae the same composition and in the same amount as for a corresponding experimental samples (equal to the addition of yeast or equal to the number of yeast cells from a dense sludge). In each control tube containing yeast cells were made with 0.9% sodium chloride solution in volume equal to the volume of solution sample CNT deposited into the appropriate test tube. Visual analysis of each of the experimental samples COP one of the samples nst item preparation) produced twice in comparison with the corresponding control sample: estimated color content of the test and control tubes directly after preparing the test and control samples and after keeping these samples in the conditions stipulated by the relevant variant of cooking KS. When setting the experiments with variation of temperature, which was maintained KS CNT samples used refrigerator pharmacological brand HF-250 "moreover, the enterprise-574" production "Incom center "Option", JSC, Russia /St. Petersburg/) and ultraharmonic "UT-15" (manufactured by Pilot plant laboratory medical equipment, Russia /Ukraine), providing for the setting of required temperature (respectively, cure for cold or temperature control).

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

The results of visual analysis of KS CNT samples with different concentrations of yeast, the amount of insertion of the CNT solution, time and temperature of aging COP selectively provided in table 1-3.

Table 1

Compliance samples narasinga of tetrazole /PCT/ proposed measure (presence of pink colouring mixture control /KS/) in terms of guaranteed adequate expert who estimates
The number of experimentsThe number of variant cooking KSThe total number of samples tested PCTThe number of samples CNT:
relevant to the proposed measurenot relevant the proposed measure
absed%absed%absed%
12345678
I110100,0880,0220,0
210100,0880,0220,0
II13100,0--3100,0
23100,0--3100,0
III13100,0133,3266,7
23 100,0133,3266,7
IV13100,0133,3266,7
23100,0133,3266,7

td align="center"> %
Table 2

The dependence of visual distinctiveness pink staining control mix /CS/ samples narasinga of tetrazole /PCT/corresponding to the proposed measure (positive samples nst), on the concentration of the yeast Saccharomyces cerevisiae, the amount of insertion of the CNT solution and time-keeping COP at a temperature of 20°
The number of positive samples investigated PCTThe number of variant cooking KSVariable parameter KSConstants KSThe total number of experts who participated in the testsThe number of experts revealed the presence of pink colouring KS
name, unit of measurename, unit of measurename, unit of measurepeoplepeople%
123456789
103the concentration of yeast, mg yeast/ml PCTthe amount of insertion of the CNT solution, mlaging time in CA, min
50,1860100,01321,7
250,1860100,03761,7
450,1860100,04981,7
500,1860100,060100,0
1500,1860100,060100,0
2500,1860100,0/td> 60100,0
2550,1860100,05083,3
2750,1860100,04371,7
3000,1860100,02541,7

Continued table 2
123456789
104the concentration of yeast, ×108yeast cells/ml of HCTthe amount of insertion of the CNT solution, mlaging time in CA, min
0,250,1860100,01525,0
1,250,1860100,03965,0
2,25 0,1860100,05286,7
2,500,1860100,060100,0
7,500,1860100,060100,0
12,500,1860100,060100,0
was 12.750,1860100,05998,3
of 13.750,1860100,05795,0
15.00,1860100,05490,0
105the amount of insertion solution HCT, mlthe concentration of yeast, mg yeast/ml PCTaging time in CA, min
0,01150860100,035,0
0,025150860100,0 2745,0
0,05150860100,060100,0
0,075150860100,060100,0
0,10150860100,060100,0
0,125150860100,060100,0
0,15150860100,060100,0
0,20150860100,060100,0

Continued table 2
123456789
106the amount of insertion of the CNT solution, mlthe concentration of yeast ×108yeast cells/ml of HCTaging time in CA, min
0,01860100,046,7
0,0257,50860100,02846,7
0,057.50860100,060100,0
0,0757,50860100,060100,0
0,107,50860100,060100,0
0,1257,50860100,060100,0
0,157,50860100,060100,0
0,207,50860100,060100,0
107aging time in CA, minthe concentration of yeast, mg yeast/ml PCT
11500,160100,02440,0
31500,160100,04473,3
51500,160100,05185,0
61500,160100,060100,0
81500,160100,060100,0
101500,160100,060100,0
121500,160100,060100,0
151500,160100,060/td> 100,0

The end of table 2
123456789
108aging time in CA, minthe concentration of yeast, ×108yeast cells/ml of HCTthe amount of insertion of the CNT solution, ml
17,500,160100,02541,7
37,500,160100,04575,0
57,500,160100,05388,3
67,500,160100,060100,0
87,500,160100,060100,0
107,500,160100,060100,0
127,500,160100,060100,0
157,500,160100,060100,0

As can be seen from table 1, the proposed criterion for the suitability of the match: in the first series of experiments - 8 CNT samples of the 10 investigated; in the third and in the fourth series of experiments on one sample from the three studied (each series). In the second series of experiments the proposed measure did not meet none of the three studied samples PCT. When this identical results were obtained when using yeast as in dry form (option 1), and in the mist (option 2). According to the results of analysis of samples of CNT has been studied in the conditions I-IV series of experiments were divided into parties. The samples corresponding to the proposed criteria prigoda the spine (positive samples nst), and the samples did not comply with the proposed criterion of suitability /also referred to as negative samples PCT", BUT "PCT"/, amounted, respectively, to the party: I -, I-HO (I series); II (II series); III, III-BUT (III series); IV, IV (series IV).

Data visual analysis (in comparison with the control samples) KS CNT samples (number examined), corresponding to the proposed criteria of suitability (batch I -, III -, IV -, under conditions of varying concentrations of yeast at constant volume made of CNT solution (0.1 ml) and a constant time-keeping samples (8 min) at a temperature of 20°With (table 2: options 3 and 4) showed the following. Immediately after cooking the contents of both experienced and control tubes were: at concentrations less than 25 mg yeast/ml NBT (using yeast in dry form) or less than 1.25×108yeast cells/ml NBT (using yeast in the form of mist) - clear solution with whitish-muddy shade; at concentrations equal to or greater than 25 mg yeast/ml NBT (using yeast in dry form) or equal to or greater than 1,25×108yeast cells/ml NBT (using yeast in the form of mist) - milky white solution. After keeping under these conditions, the appearance of control samples (including color) of whom had remained unchanged, whereas in the experimental samples was observed the presence of pink colouring COP varying degrees of severity. While at concentrations less than 25 mg yeast/ml PCT or less than 1.25×108yeast cells/ml of HCT visual determination of pink color COP was extremely difficult (if the lower limit values of the investigated concentration ranges erroneous assessment of the color of the COP took place in 75,0-78.3% of cases) due to the high transparency of the investigated COP (as shown by preliminary experiments at concentrations of 3 mg of yeast/ml NBT or 0.15×108yeast cells/ml of HCT to detect the presence of pink color managed a 1.7-3.3% of experts). In the concentration ranges of 25÷45 mg yeast/ml NBT, or from 1.0 to 2.25)×108yeast cells/ml of HCT assessment color investigated the COP as rose gave from 61,7 to 86.7% of the experts. At concentrations of 50-250 mg yeast/ml NBT or (2,5-12,5)×108yeast cells/ml of HCT was adequate expert evaluation of color COP as pink, which led to the selection of the concentrations specified range as optimal. Further increase in the concentration of yeast (in ranges 255-300 mg yeast/ml NBT (or was 12.75÷15,0)×108yeast cells/ml NBT) led to the significant reduction in the percentage of experts, Oprah is elavsky the presence of pink colouring KS (respectively, 83,3-41,7% and 98.3 to 90.0%), which was caused by decrease of intensity of the specified color of the COP due to the fact that a significant portion of yeast cells were unstained.

During visual analysis (in comparison with the control samples) positive samples CNT (number examined) when used as a variable parameter of the amount of insertion of the CNT solution at a constant concentration of yeast (150 mg yeast/ml NBT or 7,50×108yeast cells/ml NBT) and constant time-keeping samples (8 min) at a temperature of 20°With (table 2: options 5 and 6), the following is established. Immediately after cooking the contents of both experienced and control tubes was a petty-white solution (different transparency). After keeping in the specified conditions: a control sample is unchanged; in the experimental samples is the presence of pink colouring COP varying degrees of severity. When small volumes are made of CNT solution (0.01 to 0,025 ml) was relatively high error rates in visual assessment of the colour of the constitutional court, as when using yeast in a dry form and in the form of mist (respectively, 95,0-55,0% 93,3-53,3%), due to the presence of in vitro Nesmachny (and, therefore, unpainted) dry yeast or high transparency and small total volume of CA (in the case of ISOE is isawanya suspended yeast). The interval of 0.05-0.1 ml of insertion amount of the CNT solution was selected as optimal, because at the specified values of the investigated parameter was adequate expert evaluation of color COP as pink under optimal consumption of expensive chemical reagent - CNT (recommended methods for determining fat the solution flow rate PCT /at a concentration of 0.075-0.1%of the/usually does not exceed 0.1 ml). The increase in insertion solution of NCT of more than 0.1 ml did not change the accuracy of visual assessment of color COP (adequate expert evaluation pink staining), however, led to an unjustified increase in the consumption of costly solution nst.

Visual analysis (in comparison with the control samples) in terms of varying the time of incubation of the samples at a temperature of 20°With constant yeast concentration (150 mg yeast/ml NBT or 7,50×10 yeast cells/ml NBT) and constant volume (0.1 ml) made of CNT solution (table 2: options 7 and 8) showed the following. Immediately after cooking the contents of both experienced and control tubes had the appearance of a milky white solution. After keeping under these conditions, a control sample remained unchanged. In the experimental samples was observed the appearance of pink coloring of KS, since the first minute-keeping when relatively high percentage oshi is full of expert assessments (60% - for dry yeast, 58,3% for suspended yeast), which decreased with increasing time of incubation, comprising at 5 min, respectively, to 26.7% (dry yeast) and 25.0% (for the suspension of yeast). Adequate expert assessment of pink colouring KC registered, starting from the time-keeping 6 min (when using yeast as in dry form and in the form of mist). A further increase of the specified parameter (7-15 min) did not affect the accuracy of the expert evaluation. However, longer incubation over 10 min, according to the authors of the invention, it is impractical because it leads to unproductive working time of the doctor and the laboratory conducting the analysis.

The results of visual analysis (in comparison with the control samples) COP positive samples nst under conditions of varying temperature and time of incubation of the samples at each temperature in the range of variation at constant yeast concentration (150 mg yeast/ml NBT or 7,50×108yeast cells/ml NBT) and constant volume (0.1 ml) made of CNT solution (table 3: options 9 and 10) allowed us to draw the following conclusions. Immediately after cooking the contents of both experienced and control tubes had the appearance petty-white solution. After keeping under these conditions, the color samples are not visually izmenyalas experimental samples was observed appearance of pink coloring of KS, moreover, when increasing the incubation temperature to a certain critical value (22° (C) the time of occurrence of a specified color change COP was reduced, and the colour intensity is increased, leading to improved accuracy of expert evaluations; when exceeding the critical temperature (since 23° (C) the percentage of erroneous estimates were again increased. So, while keeping the COP made with yeast in dry form and in the form of a suspension, under conditions of relatively low temperatures (in the range of 2÷14° (C) adequate expert assessment of pink colouring COP became possible only at relatively long time-keeping (respectively, 20/27/÷15 min). Increasing the temperature up to 16-17°To ensure the possibility of adequate expert evaluation pink KS at an earlier time of incubation (8-10 min). And finally, in the range of 18÷22°identification of all experts (100%) of a pink colouring of the COP took place, starting in the 6th minute (the lower limit value of the interval optimal temperatures). Further increase in temperature aging COP (in the range of 23÷37° (C) resulted in drastic reduction of the time of occurrence of intense pink color COP (from 32°With the greatest accuracy of expert assessments was noted at 2-3-minute incubation), who, however, at the same time adequate expert assessment of the availability of this fact has become impossible due to the Express of the gas in the studied samples - education white foam in the amount of CA (a similar phenomenon was observed under these conditions and in the control samples). Thus, as the optimal was chosen temperature range aging COP 18÷22°C.

Comparative analysis of the results of visual analysis of the COP positive samples nst different cooking options in terms of variation of the studied parameters (table 2, 3) showed that in embodiments using a suspension of yeast at varying concentrations of yeast, the amount of insertion of the CNT solution and time-keeping of the COP at the optimum temperature obtained slightly (not statistically significant) higher results in comparison with the variants, which were dry yeast. However, under conditions of varying temperature aging COP (when using temperatures higher than the upper limit of the interval optimal values) when the optimal values of the other parameters (concentration of yeast, the amount of insertion solution nst) is less (statistically insignificant) percentage of erroneous expert assessments was recorded in variants with application of dry yeast. When the optimal values of the declared parameters of the results obtained when using dry yeast and suspended yeast, were identical. Thus, the analysis proved the validity of the sing in the formulation of NBT-test declared by way of yeast in dry form, and in the form of mist.

Before the beginning of the second phase of testing to identify the range of normal values FAF inventors have conducted researches NBT-test according to the method of the claimed method (with a parallel production test using three positive samples PCT from the party I -) 63 apparently healthy children aged from 7 days to 15 years. In a group of apparently healthy children were included, of which neither in history nor in the survey during preventive examinations on the basis of the outpatient unit No. 41 children's hospital No.1 of various clinical, instrumental and laboratory methods of variance in health status have been identified (hereinafter "healthy" children/. As a result of these studies it was found that in the formulation of NBT-test declared by way fat normal averages (27,3±3,2) formazan-positive phagocytes in a smear. The obtained values FAF normal correlate with the data of the authors of the prototype method [2].

The second phase tests were conducted comparative studies FAF defined in the parallel setting NBT-test the claimed method using all investigated in the first stage CNT samples (samples parties I -, III -, IV-HO, I-HO, II-HO, III-HO, IV-HO) had the following children:

- in children with confirmed the clinical-l is the responsibility examination impaired phagocytosis in age from 7 days to 15 years 67 people (selected from among patients hospitalized for various Department of children's hospital No.1);

"practically healthy children aged from 7 days to 15 years - 30 people (selected during preventive examinations on the basis of the outpatient unit No. 41 children's hospital No.1 before routine vaccination).

The distribution of children in the groups were as follows:

1st group -patients with confirmed low fat (7 people), including patients with recurrent furunculosis (5 people), patients with chronic granulomatous disease (2 people);
2nd group -patients with confirmed elevated fat (30 people), including patients with acute pneumonia (17 people), patients with acute intestinal infection (8 persons), patients with acute viral respiratory infections /AVRI/ (5 people);
Group 3-"practically healthy" children with FAP within normal values (30 people).

In the course of determining fat each of the surveyed children were taken by 1.9 ml of blood from a vein and contributed 0.1 ml in 19 test tubes containing each 0.1 ml pre-filled heparin (2 DB heparin in 0.1 ml). Prepared Seth. In each of the 19 test tubes containing heparinised blood specific surveyed child, was brought to 0.1 ml of pre-ol is prepared solution (from part, remaining after the first stage of testing) one of the CNT samples (solution of each of the samples in a separate test tube). The above procedure was repeated in the blood of each of the subject child. Incubated Seth (in each of the tubes) for 20 min at 37 ° °C. Then centrifuged specified mixture of each of the test tubes) for 5 min at 750 rpm Smears prepared from the precipitate obtained after centrifugation Seth (2-3 smear from each tube), after drying, fixation and staining according to the method of the claimed method were studied under the microscope in the immersion system and determined the percentage of formazan-positive phagocytes (200 cells). Assessment FAF made by comparing the obtained values with the norm according to the method of the claimed method.

Statistical processing of the results obtained in the second stage of tests was performed using criteria Fisher's exact and Wilcoxon-Mann-Whitney [6].

In table 4, 5 presents the comparative results of determination fat in the formulation of NBT-test using all studied in the first phase samples PCT in children with evidence of impaired phagocytosis and "healthy" children. In table 4 are average fat in each of the subject groups for each batch of samples of the PCT, as in table 5 - distribution is their children in each group depending on the status of the received determination results FAF (confirmed or unconfirmed).

From the data of table 4, 5 shows that the results obtained in the formulation of NBT-test the claimed method (using positive samples nst), in all groups surveyed children fully correlated (direction and relative compared to the norm quantitative values) with confirmed (with prior clinical and laboratory examination) functional parameters of the system of phagocytosis. The use of negative samples nst allowed to obtain formal coincidence of the direction of the disturbances in the system of phagocytosis only in patients with confirmed clinical and laboratory data of the reduced fat (1st group). However, the quantitative values of fat (reduction 96,7-98,5% compared to the norm) did not correspond to the same values obtained using both pre-clinical and laboratory examination, and the use of positive samples nst (reduction 73,6 is 74.7% compared to the norm). In patients with confirmed elevated PAF (2nd group) and apparently healthy children (group 3) when using the negative samples nst all results determine fat was loginusername in 100% of cases. In both of these groups received a reduction fat compared snormal: in the 2-nd group - by 95.9-96,3% (confirmed result - increase fat on 68,1-75,1% compared to the norm); the 3rd group - by 95.9-96,6% (confirmed result is in the range of normal values).

To prove the specificity used in the proposed method the test culture (Saccharomyces cerevisiae) were comparative experimental studies concerning the presence or absence of pink colouring COP three positive samples CNT from party 1-/further - HCT-1", "PCT-2", "PCT-3"/prepared for each of these samples nst) using as initial components of yeast cells and leukocytes in various concentrations. Was previously prepared in three test tubes with a volume of 10 ml (each) 0.1% solution of each of the three studied in this series of experiments positive samples CNT (CNT-1, CNT-2, NCT-3)dissolved in 0.9% solution of sodium chloride. Then prepared according to the method of the claimed method, the COP of each of these samples PCT /hereinafter - "the COP nst-1", "KS nst-2", "KS nst-3"/ using as a source component in a suspension of the yeast Saccharomyces cerevisiae. To obtain the suspension of yeast used a strain of Saccharomyces cerevisiae grown on solid nutrient medium Saburo. Grown yeast washed three times in 0.9% sodium chloride solution according to the claimed method. The concentration of yeast cells in the volume density is th sediment (obtained after removal of the supernatant liquid after the last centrifugation) was 2.5× 1012cells/ml thick sludge. Made cages of a dense sludge 12 transparent centrifuge tubes (No. 1-12) in the following number /next - experienced test tube "D"/:

in test tube No. 1, 5, 9 - to 0.0001 ml thick sludge;

in test tube No. 2, 6, 10 - 0,0003 ml thick sludge;

in test tube No. 3, 7, 11 - 0.0005 ml thick sludge;

in test tube No. 4, 8, 12 - 0,0006 ml thick sludge.

Then selected four portions of each of the previously prepared solutions of samples nst-1, PCT-2 PCT-3 with a volume of 0.1 ml (each portion) and added these portions in test tubes (No. 1-12)containing yeast cells from a dense precipitate (in each of the grouped according to the four tubes /No. 1-4; 5-8; 9-12/ - on one portion of one of the samples PCT /respectively, CNT-1, CNT-2, NCT-3/). The concentration of CA nst-1 in test tubes No. 1-4; CA nst-2 in test tubes No. 5-8, CA nst-3 in test tubes No. 9-12 amounted to, respectively: (a 2.5×108), (7,5×108), (12,5×108) and (15,0×108) the yeast cells/ml of the appropriate sample PCT /hereinafter - "pilot test "D"/. In parallel with each of the experimental samples "D" in the same tubes (hereinafter "control tube "D"/ prepared control sample (hereinafter "control sample "D"/. At the same time as the source component for the preparation of each control sample "D" used the yeast Saccharomyces cerevisie in the same amount, as for corresponding to the experimental sample "D" (equal to the number of yeast cells from a dense sludge). In each control tube "D"containing yeast cells were made with 0.9% sodium chloride solution in volume equal to the volume of solution sample CNT deposited into the appropriate test tube "D". Kept for each of the experimental and control tubes "D" for 8 min at a temperature of 20° (average values stated interval optimal values of these parameters). Visual analysis of each of the experimental samples "D" was produced twice in comparison with the corresponding control sample "D": to evaluate the color content of the test and control tubes "D" directly after preparation of the test and control samples "D" and after keeping the data samples in the specified conditions.

Prepared KS samples CNT-1, CNT-2 and HCT using as a source component in a suspension of cells. The plasma-containing blood leukocytes were obtained by the method described in [7]. However, heparinized blood (20 U/ml) was added 10% gelatin (10 ml of whole blood - 1 ml of 10% gelatin). Put 30 minutes in a thermostat at 37°C. the Plasma with leukocytes was sucked out Pasteur pipette and used for further studies. The leukocytes were washed from the plasma three times in 0.9% sodium chloride solution by centrifuge the simulation at 1500 rpm for 15 minutes After the last centrifugation the supernatant was aspirated Pasteur pipette. In the amount of dense precipitate obtained after removal of the supernatant liquid was determined by the concentration of leukocytes, which amounted to 2.5×1011cells/ml thick sludge. Made cages of a dense sludge 12 centrifuge tubes (No. 13-24) in the following number /next - experienced test tube "L"/:

in test tube No. 13, 17, 21 - 0,001 ml thick sludge;

in test tube No. 14, 18, 22 - 0.003 ml thick sludge;

in test tube No. 15, 19, 23 - 0,005 ml thick sludge;

in test tube No. 16, 20, 24 - 0,006 ml thick sludge.

Then selected four portions of each of the previously prepared solutions of samples nst-1, PCT-2 PCT-3 with a volume of 0.1 ml (each portion) and added these portions in test tubes (nos 13-24)containing leukocytes from a dense precipitate (in each of the grouped according to the four tubes /No. 13-16; 17-20; 21-24/ - on one portion of one of the samples PCT /respectively, CNT-1, CNT-2, NCT-3/). The concentration of CA nst-1 in test tubes No. 13-16; KS nst-2 in test tubes No. 17-20, CA nst-3 in test tubes No. 21-24 amounted to, respectively: (a 2.5×108), (7,5×108), (12,5×108) and (15,0×108) leukocytes/ml of the appropriate sample PCT /hereinafter - "pilot test "L"/. The maximum of the studied concentrations (15,0× 10)8leukocytes/ml of HCT) was determined by the highest number of cells that were able to isolate from the blood plasma of the allowable blood volume, which could be selected from the surveyed child without prejudice to the state of his body. In parallel with each of the experimental samples "L" in the same tubes (hereinafter "control tube "L"/ prepared control sample (hereinafter "control sample "L"/. At the same time as the source component for the preparation of each control sample "L" used leukocytes in the same amount as for a corresponding experimental samples "L" (equal to the number of cells of the dense sludge). Further preparation of the reference sample "L" was performed similarly to that described above for the reference sample "D". Kept for each of the experimental and control tubes "L" in the same conditions, a test tube "D" (8 min at a temperature of 20°). Visual analysis was made similar to that described above for the experimental samples "D".

Visual analysis of the COP, yeast and cells in various concentrations (respectively, experienced a test tube "D" No. 1-12, power tubes "L" No. 13-24), produced using the method of expert evaluation, as described above for the experiments of the first phase of testing (with the participation of 60 experts). The analysis established the presence of Il is the lack of pink staining of CA in each of the studied experimental tubes ("D" and "L") after storage in the stated conditions.

In table 6 presents the results of a comparative visual analysis of the COP prepared using as starting components of yeast cells and leukocytes in various concentrations (selectively - for example, the COP HCT-1).

Table 6

A comparative study of the possibilities of the visual assessment of response recovery narasinga of tetrazole /PCT/ formazan when using the mixture control positive sample CNT-1 /CA nst-1/containing yeast cells and the cells in various concentrations
Type of cells used as starting components for the preparation of the COP nst-1Rooms tubes containing CA nst-1The concentration of CA nst-1×108cells/ml NBT-1The number of experts revealed the presence of pink colouring KS CNT-1
people%
12345
Yeast cells12,560100,0
27,560100,0
312,560100,0
4 15,060100,0
Leukocytes132,500
147,500
1512,500
1615,000

The analysis of the results of visual analysis of the COP studied the positive samples HCT - KS nst-1 (data 6), KS nst-2 and KS CNT-3) showed the following. Immediately after cooking the contents of the test and control tubes "D" was a milky white solution, and the contents of the test and control tubes "L" - solution with whitish-opaque shade different transparency depending on the concentration of cells in solution. After keeping under these conditions in all experimental samples "D" (for all the investigated concentrations of yeast cells for all tested samples nst) all experts (100%) reported the presence of pink color COP. At the same time, when the use of cells in all experimental samples "L" (for all the investigated concentrations of cells for all tested samples PCT) after a similar incubation took place adequate assessment of absence of pink colouring KS (color content of all the op is the shaft tubes "L" was identical to the color content of the respective control tubes "L").

Achievement provided by the invention technical result is the following. It is known that phagocytophilia microorganisms (bacteria, viruses, fungi, protozoa) dissolved in phagocytes with the help of various enzymes, including oxidase free nucleotides OVER·H and NADP·N [2, 7]. It is established that many living cells (mainly having a nucleus capable of capturing a drop in the surrounding liquid. This phenomenon is called pinocytosis [8]. When setting NBT-test methods a method similar to [1]and the prototype method [2] after incubation with a solution of HCT in the cytoplasm functionally active faguoqitirute cells observed the formation of granules formazan violet-blue [1] or blue [2] colors that can be seen under microscopy with oil immersion magnification. Given that these methods of setting NBT-test reaction for reduction of NBT in the insoluble pellet formazan occurs in phagocytes (white blood cells) in the absence of corpuscular objects phagocytosis, the penetration of the PCT in phagocytes is, according to the authors of the invention, by pinocytosis.

Yeast cells do not traditionally belong to the phagocytes. However, the results of experimental researches of the authors of the claimed method showed that after incubation with a solution of HCT in yeast cells is determined (under Immers the pension increase) is very small, dusty pink inclusions, diffusely distributed in the volume of cells. These include, according to the authors of the proposed method, are granules formazan formed in the restoration CNT. Pink (not purple-blue or blue) coloration of these granules, apparently, due to the smaller (compared to phagocytes) the size of these granules and, accordingly, the higher the transparency. Thus, the obtained experimental data suggest that yeast cells contain (as well as phagocytes) oxidase free nucleotides OVER·H and NADP·N, is able to restore the PCT in formazan. The formation of granules formazan in yeast cells in the presence of CNT, according to the authors of the invention, also occurs as a result of pinocytosis solution specified chromogenic reagent. Experimental data of the authors of the invention have established that incubation mixtures containing yeast cells in a solution of CNT /CC/in some cases (when using a number of samples of HCT, under certain parameters KS and conditions of incubation) resulted visually (macroscopically) distinct pink coloration of the COP. In the trial, which saw the emergence of macroscopically distinguishable pink KS, microscopy under oil immersion magnification noted ALOS education granules formazan pink testified to the positive result of the reduction reaction of the PCT. Absence (with the same parameters KC and the conditions of incubation, but when using other samples nst) of the specified color change COP in the samples during visual analysis correlated with the total absence of pink granules formazan with microscopy, which indicated that the recovery of the CNT in these samples did not happen. The appearance of macroscopically distinguishable color COP with a positive result of the reduction reaction of the PCT, according to the authors of the invention, due to the fact that the granules formosana are formed in the surface layers of yeast cells (layers of cytoplasm adjacent to the cell membrane or in the cell layers within the structure of the membrane) and/or relatively high transparency membranes of yeast cells that, in turn, leads to the formation of color COP sufficiently intense to visually distinguish. Experimental studies of the inventors also showed that after incubation (at optimum values of the declared parameters) mixtures containing functionally active faguoqitirute cells (leukocytes) in the solution of the positive sample nst, even at relatively high concentrations of leukocytes in the above-mentioned mixture visually distinct pink color of the latter was not observed (nesm is rubbing on the presence of granules formazan, determined under oil immersion magnification). According to the authors of the proposed method, this may be due to the fact that in leukocyte granules formosana are formed in the deeper cell layers (layers remote from the cell membrane) and/or lesser transparency of these phagocytose cells.

Thus, it is the structural and functional characteristics of the membranes and the surface layers of the cytoplasm of the used test cell culture provide the ability to pre-macroscopic assess the ability of the PCT to restore formazan. This allows pre - (before setting NBT-test) to assess the suitability of a particular sample CNT for use in the reaction of phagocytosis, which ensures the reliability of the results of the NBT-test and, thereby, increases the accuracy of determining fat.

In experimentally selected interval, the optimal values of concentrations, KC is achieved is the ratio between the number of yeast cells and the quantity of applied chromogenic reagent - CNT, which is necessary and sufficient to cause the formation of formazan-positive cells in a quantity sufficient to cause coloration of the COP of such intensity, which is adequate physiological resolution of the organ of vision of a person (hereinafter referred to as "physiologically the Ki adequate intensity of staining KS/, which makes the possibility of adequate expert evaluation of color COP as pink. The use concentrations of the COP outside the lower and upper bounds of the interval of the optimal value leads to the formation of formazan-positive cells in a quantity sufficient to provide physiologically adequate intensity of staining KS (intensity of staining KS - beyond the physiological limits of the resolution of the organ of vision), in the first case due to the lack of yeast cells in relation to the PCT, and in the second case because of their abundance. This complicates or makes it impossible for the visual determination of the existence of pink colouring of the COP and, as a consequence, leads to the impossibility of adequate expert assessment of this fact.

The optimal amount made solution nst (not less than 0.05 ml), on the one hand, allows to obtain an optimal ratio between the number of yeast cells and the number of deposited reagent (BAT), which leads to a physiologically adequate intensity of the pink colouring of the COP and, accordingly, creates conditions for adequate expert evaluation of the specified color COP. On the other hand, using the optimal values of the specified parameter (not more than 0.1 ml) avoids unnecessary consumption of expensive chromogenic reagent - the ARTICLE (added advantage).

The optimum temperature and time parameters of incubation KS contribute the most to the rapid emergence of a physiologically adequate intensity of staining COP with the exception of the possible negative impact of physical factors that hinder visual assessment of color COP. During curing of the COP in terms of temperatures that are outside the lower limit of the interval of the optimal values of this parameter (less than 18° (C)increases the time required to complete the reduction reaction of CNT in yeast cells, with the formation of granules formazan in an amount to provide the appearance of a physiologically adequate intensity of staining of the COP and, accordingly, the time when the results of the expert evaluation pink KS be adequate. During incubation the COP at temperatures higher than the upper bound of the range of optimal values (>22° (C)reduces the time required for the formation of granules formazan in a quantity sufficient to achieve a physiologically adequate intensity pink colouring KS. However, this dramatically increases the formation of yeast cells carbon dioxide (CO2), which leads to the formation of white foam in the amount of CA in the very first minutes of incubation, which, in turn, eliminates the possibility of adequate expert evaluation pink CEE is and CS at any time-keeping, including at the optimum. Under optimal temperatures using the optimal values of time of incubation KS (6-10 min) ensure adequate expert evaluation of the pink colouring of the COP (at time of at least 6 min) with the exception (if time is not more than 10 minutes) the possibility of non-productive working time of staff immunological laboratories participating in the formulation of the NBT-test (added advantage).

Additional centrifugation Seth during production, NBT-test (in particular, for 5 min at 750 rpm) allows you to concentrate blood cells, including phagocytes (due to the removal of the liquid portion Seth), and, thereby, reduce the time spent on defining fat, in particular for counting in a smear formazan-positive phagocytes among all of phagocytes (added advantage).

Using the stated intervals the optimal values of concentrations, KC, of the amounts made solution nst, as well as temperature and time of incubation parameters enables equal application of yeast cells regardless of their functional status that extends the capabilities of the claimed method. According to the authors of the invention, yeast are mainly in a functionally inactive state, and to restore their functional SV is Istv when placed in a solution of CNT take some time. In suspensions of yeast cells are functionally active and when declared breeding retain their functional properties at the same level. Accordingly, the use of suspended yeast by varying these parameters incubation outside the interval optimal values led to faster reaction recovery of HCT in the cells (with the advent in the physiologically adequate intensity of staining COP) at lower temperatures and at lower concentrations of CA than in the case of using dry yeast. However, at high temperatures (at the upper boundary of the interval of the optimal value) higher intrinsic functional activity of yeast cells in suspension (compared to dry) resulted in higher gas production, which reduced the accuracy of visual assessment and excluded the possibility of adequate expert evaluation of color COP. Within the stated intervals the optimal values of the parameters of the incubation, the COP was adequate expert evaluation of color COP regardless of the functional state of the used yeast cells, suggesting a possible use in the claimed process as dry yeast, and the suspension of yeast at the same validity of the obtained results.

The way the implementation is given as follows. Pre-cooked, such as in vitro displacement of more than 0.2 ml of 0.1% solution of NBT dissolved in a solution of sodium chloride isotonic 0.9%. Prepare COP working concentration for which part of a pre-prepared CNT solution is mixed with an appropriate amount of yeast Saccharomyces cerevisiae. When using as the source component for the preparation of the CC of dry yeast corresponding to a portion of the yeast is placed in a transparent tube, such as the centrifuge. Take, for example a metered dropper part of a pre-prepared solution of CNT volume of 0.05-0.1 ml and applied to a test tube containing a sample of yeast. The mass of a sample is determined depending on the specific scope of proposed solution the PCT in order to provide a working concentration of CA 50-250 mg yeast/ml NBT. When using as a source component suspended yeast COP is prepared as follows. To obtain suspensions using strains of Saccharomyces cerevisiae grown, for example, on a dense nutrient medium Saburo. Grown yeast wash with 0.9% sodium chloride solution and then washed three times in 0.9% sodium chloride solution by centrifugation at 3000 rpm for 15 min After the last centrifugation the supernatant is sucked off, such as the Pasteur pipette. In the dense volume is the charge, obtained after removal of the supernatant liquid, determine the concentration of yeast cells (hereinafter "initial concentration of yeast cells"/. Take, for example, using automatic metered micropipette, a corresponding number of cells of the dense sludge and make them transparent tube, such as the centrifuge. Take, for example a metered dropper part of a pre-prepared solution of CNT volume of 0.05-0.1 ml and introduced into the test tube with yeast cells. The quantity of yeast cells is determined depending on the initial concentration of yeast cells and a specific amount of insertion of the PCT in order to provide a working concentration of CA (2,5-12,5)×108yeast cells/ml NBT. Bear COP for 6-10 minutes at a temperature of (20±2)°C, after which produce a visual analysis of its color. If the COP pink colouring the rest of the pre-prepared solution of HCT used for cooking Seth. Produce blood with anticoagulant in the subject. In silikonizirovannaya centrifuge tube containing 0.1 ml pre-filled heparin (2 UNITS of heparin in 0.1 ml), contribute 0.1 ml of blood taken from a finger, and gently mixed with heparin. Prepare Seth. In silikonizirovannaya centrifuge tube, soda is containing heparinised blood, contribute 0.1 ml of the previously prepared solution of HCT (remaining part). Incubated Seth for 20 min at 37 ° °With (e.g., thermostat). Optimally after incubation to produce centrifugation Seth, for example (as provided by the method of the method-analogue of [1]) for 5 min at 750 rpm From the contents of the tubes obtained after incubation Seth, or from the precipitate obtained after centrifugation Seth (if the latter was made), prepare smears for low-fat slides (2-3 smear from each tube /blood/). Prepared smears air-dried, and then fixed by one of known methods, for example in a mixture of ethanol:formalin (in the ratio 9:1) for 30 s [1] or in absolute methyl alcohol for 10 min [9]. Then paint strokes (one of the known techniques) dye providing selective coloring cell nuclei, for example (as provided in the method-analogue [1]) 0.1% solution of neutral red dissolved in 0.5% solution of sodium chloride. After staining nuclei strokes are viewing under the microscope in the immersion system: on each of the strokes is considered to be not less than 200 cells (total number of phagocytes) and from them determine the percentage of formazan-positive phagocytes. Calculate the average percentage of formazan-positive phagocytes in smear is. The obtained value of the specified indicator is correlated with the norm, which, according to the authors of the invention is (27,3±3,2) % formazan-positive phagocytes in a smear. The matching results establish the presence or absence of changes of the studied parameters in comparison with the norm. When the metric value is within normal limits define normal fat, with a decrease compared with standard - lower fat, while increasing the value of the indicator compared with standard - higher fat.

The invention is illustrated by the following examples.

Example 1.

Boy Dima S., age 6. Delivered accompanied by a parent in the outpatient Department No. 41 children's hospital No.1 for permission for routine vaccination against polio. Complaints the child and the parents were not charged. The child from normal pregnancy, emergency childbirth; the neonatal period without pathology. History of the child rare (1-2 times per year) AVRI. Experts are not observed. Allergic diseases are not present. Previous vaccination without complications. The child examined by a pediatrician. Diagnosis: healthy. Contraindications to vaccination no.

At the request of parents was a comprehensive examination of the child with the use of various clinical, instrumental and laboratory methods, including defined the e FAF using known techniques. In addition, the definition FAF also manufactured using the inventive method.

According to the method of the claimed method was pre-test two samples of CNT obtained from different sources /further - PCT-To" and "HCT-L"/, according to the proposed measure. During this test previously in two test tubes with a volume of 1.5 ml each were prepared in 0.3 ml of 0.1% solution of each of the test samples (PCT and HCT-L), dissolved in 0.9% solution of sodium chloride. To prepare the COP of each of the test samples (PCT and HCT-L) using Saccharomyces cerevisiae in a dry form or in the form of mist. When using as a source component dry yeast two hinge yeast mass by 2.5 mg each, were placed in two transparent centrifuge tubes with a volume of 10 ml Metered dropper chose to 0.05 ml of the previously prepared solution of each of the samples nst and introduced into a test tube containing a sample of yeast (each CNT sample in a separate test tube). The working concentration of CA in each of these tubes was 50 mg yeast/ml solution of the appropriate sample PCT /then "test tube No. 1K and 1L"/. Directly after preparation of the COP in test tubes No. 1K and 1L had a milky-white color. When using as a source component in a suspension of yeast every COP is on of the test specimens CNT was prepared as follows. Used a strain of Saccharomyces cerevisiae grown on solid nutrient medium Saburo. Grown culture of yeast was washed with 0.9% solution of sodium chloride and then washed three times in 0.9% sodium chloride solution by centrifugation at 3000 rpm for 15 min After the last centrifugation the supernatant was aspirated using a Pasteur pipette. In the amount of dense precipitate obtained after removal of the supernatant liquid was determined by the initial concentration of yeast cells, which amounted to 2.5×1012cells/ml thick sludge. Selected with the help of automatic metered micropipette on 0,0000125 ml thick sludge and made of two transparent centrifuge tubes with a volume of 10 ml Metered dropper chose to 0.05 ml of the previously prepared solution of each of the samples nst and made into tubes with yeast cells (each CNT sample in a separate test tube). The working concentration of CA in each of these tubes was 2.5×108yeast cells/ml of the appropriate sample PCT /then "test tube No. 2K and 2L/. Directly after preparation of the COP in test tubes No. 2K and 2L had a milky-white color. Withstood the test tube No. 1R, 1L, 2R and 2L, containing the KS test specimens PCT for 6 minutes at a temperature of 18°C, after which he made a visual analysis of the ROC the ski COP in each of the investigated tubes. In test tubes No. 1K and 2K noted the presence of strong pink coloring of KS, which allowed us to make a conclusion on compliance of the sample nst-To the proposed measure and, consequently, the possibility of using this sample for preparation of Seth in the course of producing NBT-test. The contents of the test tubes No. 1L and 2L after keeping under these conditions had a milky-white color, indicating that the mismatch of the sample HCT-L the proposed measure and, consequently, inappropriate use of this model for setting NBT-test.

The examined child from a finger picked 0.1 ml of blood, put it in silikonizirovannaya centrifuge tube containing 0.1 ml pre-filled heparin (2 UNITS of heparin in 0.1 ml), and gently mixed the blood with heparin. Prepared Seth. In silikonizirovannaya centrifuge tube containing heparinised blood, made 0.1 ml of the previously prepared solution of CNT sample (remaining part). He plaincourault Seth for 20 min at 37 ° ° (thermostat). Was ofcentrifugal Seth for 5 min at 750 rpm From the precipitate obtained after centrifugation Seth, had prepared three stroke low fat slides, dried in air, and then were fixed in a mixture of ethanol:formalin (in which the rate of 9:1) for 30 s Smears were stained with 0.1% solution of neutral red dissolved in 0.5% solution of sodium chloride (for staining of nuclei). After staining nuclei smears were viewed under the microscope in the immersion system: on each smear was considered not less than 200 cells (total number of phagocytes) and one of them was determined by the percentage of formazan-positive phagocytes. Calculated the average percentage of formazan-positive phagocytes in a smear, which amounted to 27.9%. Comparison with the norm allowed to determine the examined child normal fat.

In the course of a comprehensive clinical and laboratory examination of the patient revealed the following. At clinical examination of deviations from internal organs is not found.

The blood formula - without a pathology.

In the immunological (relative and absolute number of T - and b-lymphocytes; the concentration of immunoglobulins a, M, G; the content of components of the complement system; spontaneous and induced cytokine production; the level of circulating immune complexes (CEC); chemotaxis of neutrophils; indicators FAF) abnormalities not detected. In particular, the indicators FAF defined according to the technique described in the patent of the Russian Federation No. 2242763 [9], was: FI=93% at the rate 94,18%±1,55%); FC=10,9 (at the rate of 11.29±1,00); CFC=1,18 (at the rate of 1.16±0,04); FI=94% (the norm 92,00%±2,52%); FC=92 (the norm 9,81± 0,96); BIN=68% (the norm to 66.30%±2,58%). The values of all these parameters of phagocytosis in normal limits.

Thus, the results of the comprehensive examination showed the absence of disturbances in the system of phagocytosis. The results determine fat obtained by the claimed process were consistent with results obtained by other methods in the framework of the comprehensive examination.

Example 2.

Patient Nastya Century, 7 years. Delivered in the outpatient Department No. 41 children's hospital No.1 with complaints of cough and fever up to 39°C. Sick for three days. Received symptomatic therapy (fever and expectorants), but the condition did not improve, and therefore appealed to children's hospital No.1. History of the child rare (1-3 times per year) AVRI. Experts are not observed. Heredity is not burdened.

When viewed from the child's pediatrician condition regarded as moderate: fever up to 38.5°S, small shortness of breath, mild perioral cyanosis. Percussion of the chest revealed dullness of percussion sound over the lower lobes of the right lung; here auscultation found the weakening of breathing, traiterous wheezing. From other internal organs revealed no pathology. On the chest x-ray detected infiltration in the lower lobe of the right lung. On the basis of the AI clinical and radiological findings diagnosed with Community-acquired acute right-sided inferior pneumonia. On the same day the girl was hospitalized on somatic branch №7 children's hospital No. 1, and she was given a comprehensive clinical and laboratory examination, including determination FAF known methods. In addition, it was the comparative definition FAF using the prototype method [2] and the claimed method.

In connection with the temporary absence of the immunological laboratory as dry yeast, and culture of Saccharomyces cerevisiae, was first delivered nst-test according to the method of the prototype method [2] using one of the available immunological laboratory samples PCT /hereinafter - "nst-M/ without a preliminary assessment of its suitability. According to the method prototype [2] have examined the child made the blood in a test tube with heparin analogously to example 1. Prepared Seth. When 0.1 ml of heparinized blood was mixed with 0.1 ml of 0.1% solution of the sample CNT-M in 0.9% sodium chloride solution. He plaincourault Seth for 20 min at 37 ° °C. From the contents of the tubes obtained after incubation Seth, had prepared three smear and made them microscopy (under oil immersion magnification). In each of the strokes has identified a number of formazan-positive phagocytes in percent of the total number of phagocytes and calculated the average percentage of formazan-positive phagocytes in a smear, which amounted to 0.7%. If the pressure from the norm [2] testified marked reduction fat (97,4% compared to the norm).

When combined clinical and laboratory examination of the patient revealed the following results.

In the analysis of blood formula revealed an increase in the number of cells to 16.7×10%; the shift in the formula left (stab 11%); increased sedimentation rate up to 32 mm/h

In the immunological identified: a slight increase in the number of b-lymphocytes (SD 25% at the rate of 11-16%); a slight increase in the concentration of immunoglobulin M (Ig M 150 mg % at the rate 24-112 mg %); a slight increase of IL-1β (spontaneous production of 53 PG/ml at the rate of 30-50 PG/ml) and IL-2 (spontaneous production of 0.65 units/ml at norm 0-0,5 units/ml); a slight increase in the chemotaxis of neutrophils (index migration under the influence of FMLP to 3.1 at the rate of 2.6 to 2.8). Other immunological parameters - the content of T-lymphocytes; the concentration of immunoglobulins A, G; the content of components of the complement system; induced cytokine production and the level of the CEC were within age norms. Indicators PAF [9] was: FI=99%; FC=17,2; FI=100%; FC=14,9; CFC=1,15; IBN=67,3%. When compared to the normal values found increased FC (52.3% compared to the norm) and FC (51.9% compared to the norm); other indicators are within normal limits.

Thus, the results of a comprehensive survey testified system activation of phagocytosis in this patient.

In connection with those is that the results of determining fat, obtained using the prototype method [2], contrary to the results obtained using other methods as part of the comprehensive clinical and laboratory examination, bacteriological laboratory received the samples of Saccharomyces cerevisiae in a dry form or in the form of the grown culture (Saccharomyces cerevisiae strain grown on solid nutrient medium Saburo), and on the same day was made repeated examination of the blood of the child in NBT-test with another image of CNT /further - HCT-N"/ according to the claimed method.

In the implementation of the claimed process was previously performed sample HCT-N according to the proposed measure. In parallel, the same compliance test was conducted and the sample CNT-M, previously used for the production NBT-test method prototype. Compliance test samples HCT and HCT-N was carried out analogously to example 1, including the preparation of a 0.1% solution of each of these samples, dissolved in 0.9% sodium chloride solution; the preparation of the COP of each of these samples using Saccharomyces cerevisiae in a dry form or in the form of mist and maintaining each of the studied COP with subsequent visual analysis of their color. When used as the initial component of dry yeast:

the mass of each of the two sub-samples drogi the th, made of two transparent centrifuge tubes to 15.0 mg;

- the volume of solution of each of the samples HCT made in tubes containing samples of yeast, 0.1 ml;

- working concentration of CA in each of these test tubes 150 mg yeast/ml solution of the appropriate sample PCT /then"test tube No. 1M and 1N"/;

the painting of the COP in test tubes No. 1M and 1N immediately after cooking - milky-white.

When using as a source component suspended yeast:

- initial concentration of yeast cells to 3.0×1012cells/ml thick sludge;

the number of yeast cells that are made in each of the two transparent centrifuge tubes, 0,000025 ml thick sludge;

- the volume of solution of each of the samples HCT made in test tubes with yeast cells, 0.1 ml;

- working concentration of CA in each of these tubes is 7.5×108yeast cells/ml of the appropriate sample PCT /then "test tube No. 2M and 2N"/;

the painting of the COP in test tubes No. 2M and 2N immediately after cooking - milky-white.

Withstood the test tube No. 1M, 1N, 2M and 2N, containing the KS test specimens PCT, for 10 minutes at a temperature of 20°C. the Results of visual analysis of color COP:

in test tubes No. 1M and 2M after keeping under these conditions, the contents had Opocno-white color, that allowed us to draw the conclusion about the unsuitability of the sample CNT-M for use in the formulation of NBT-test.

in test tubes No. 1N and 2N after aging was observed pronounced pink color COP, testified to the validity of the sample HCT-N for setting the NBT-test.

After completion of verification of conformity of the examined child made the blood in a test tube with heparin analogously to example 1.

Prepared Seth using sample HCT-N analogously to example 1. The average percentage of formazan-positive phagocytes in a smear was 41.8%, which allowed us to diagnose the child increased fat (excess by 53.1%).

Thus, the results of determining fat obtained using the inventive method, a fully correlated with data obtained using other methods due diligence, not only in the part concerning the direction of change in the system of phagocytosis, but also in part quantitatively characterize the level of the specified offset.

Example 3.

Patient Misha Century, 8 years. The parents brought the child to the receiving unit of children's hospital No. 1 with complaints of abdominal pain, fever up to 40°C. Deterioration of the child noted in the last week before treatment: increasing intensity of pain in the abdomen, poor appetite, increasing hyperthermia. A parent is considered child patients 3 years of age when the last with a similar clinical picture was admitted to the hospital; at the same time after the survey was diagnosed with liver abscess, about which the child was operated on. Since then, the boy was repeatedly hospitalized in the hospital with recurrent agentlastname soft tissue; once - over of abscess pneumonia.

Upon admission to children's hospital No.1 child was examined by the duty surgeon. Palpation of the abdomen revealed a sharp pain in the right hypochondrium, enlargement of the liver 3 see From other organs pathology was not detected. Ultrasound abdomen revealed a well-rounded education with a diameter of 4 cm from the liquid level in the liver. Suspected liver abscess.

A complex clinical and laboratory examination, including immunological examination, including determination FAF known methods. In addition, there was a definition FAF using the inventive method. Because the presence of abscesses in the liver requires differential diagnosis of echinococcosis, a comprehensive survey was also included serological testing for Echinococcus. The results of serological tests were negative, and therefore echinococcosis was excluded.

Immediately after por is conducting a comprehensive examination, the child was operated on. During surgery revealed an abscess in the right lobe of the liver.

When setting NBT-test according to the method of the claimed method pre-test was conducted selected for use (from the party received immunological laboratory) sample CNT (manufactured by ICN Pharmaceuticals, Inc.", USA) /next - "nst-R/, for compliance with the proposed measure. Compliance test sample nst-R was carried out analogously to example 1, including the preparation of a 0.1% solution of the specified sample, dissolved in 0.9% sodium chloride solution; the preparation of the COP specified pattern using Saccharomyces cerevisiae in a dry form or in the form of suspension and keeping the studied COP with subsequent visual analysis of its color. When used as the initial component of dry yeast:

the weight of the yeast, made in transparent centrifuge tube, is of 18.75 mg;

- the volume of the sample solution nst-R made in a test tube containing a sample of yeast - 0.075 ml;

- working concentration of CA in the specified test tube 250 mg yeast/ml of the sample solution nst-R /next - "test tube No. 1P"/;

the painting of the CA in vitro No. 1P immediately after cooking - milky-white. When using as a source component suspended yeast:

- initial concentration of yeast cells to 2.5×1012cells/the l dense sludge;

the number of yeast cells that are made in transparent centrifuge tube, - 0,0000375 ml thick sludge;

- volume solution sample nst-R made in a test tube with yeast cells, - 0.075 ml;

- working concentration of CA in the specified tube - 12,5×108yeast cells/ml of the sample solution nst-R /next - "test tube # 2P"/;

the painting of the CA in vitro # 2P immediately after cooking - milky-white.

Withstood the test tube No. 1P and 2P, containing the KS test sample nst-R, for 8 min at a temperature of 22°C. the Results of visual analysis of color COP:

in test tubes No. 1P and 2P after keeping in the specified conditions noted marked pink staining COP that talked about the suitability of the sample nst-R for use in the reaction of phagocytosis.

After completion of verification of conformity of the examined child made the blood in a test tube with heparin analogously to example 1. Prepared Seth using sample nst-R analogously to example 1. The average percentage of formazan-positive phagocytes in a smear was 0.8%, which indicated a reduced fat patient (down to 97.1% compared to the norm).

In the course of a comprehensive clinical and laboratory examination of the patient revealed the following.

In the analysis of blood with the formula: increase in the number leukocyte is to 18.9× 109/l; the shift in the formula left (stab 13%); increased sedimentation rate to 58 mm/h

In the immunological: increase the number of b-lymphocytes (SD 32% at the rate of 11-16%); hyperimmunoglobulinemia M and G (JgM 180 mg % at the rate 24-112 mg %; JgG 1900 mg % at the rate 583-1783 mg %); increased IL-2 (spontaneous production of 0.8 units/ml at norm 0-0,5 units/ml); increased CEC (132 units/ml at a rate of up to 120 units/ml). Other immunological parameters - the content of T-lymphocytes; the concentration of immunoglobulin a; the content of components of the complement system; induced production of cytokines; chemotaxis of neutrophils within age norms. Indicators PAF [9] was: PI=36%; FC=2,0; FI=28%; FC=2,0; KFC=0; BIN=1.8 percent. The result of the comparison with normal values revealed a significant decrease in phagocytosis compared to the norm, especially KFC (a reduction of 100%) and IBN (reduction of 97.3%).

Thus, the data of the comprehensive examination testified deep disturbances in the system of phagocytosis, in particular the sharp decline in digestive ability of phagocytes. The nature and severity (compared to the norm) changes FAF identified using the inventive method, was in full accordance with the results obtained using other methods in the framework of the comprehensive examination. In General, the results of a comprehensive survey, the Finance, in particular, the provision in the history of re-operation for recurrent liver abscesses and soft tissue in combination with diminished performance FAF, allowed the diagnosis of chronic granulomatous disease.

The claimed method was applied in the survey of 186 children aged from 7 days to 16 years, which was based on inpatient and outpatient departments (No. 41) children's hospital No.1. All children were given a comprehensive survey with application of various 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 study of chemotaxis of peripheral blood leukocytes according to methodology described in [2]; the definition FAF according to methodology described in [9]). Taking into account medical history, clinical picture and conducted comprehensive surveys were intended clinical diagnoses /hereinafter - "the results of the control of the comprehensive examination (CE)"/. While the surveyed children were divided into the following groups:

Group I -patients from group sickly and on the Tay, with a history of experienced frequent (more than five times per year) incidence OVRI (57 people);
Group II -patients with acute pneumonia and bronchitis (22 people);
Group III -patients with acute intestinal infections (17 people);
Group IV -patients with chronic gastritis, gastroduodenitis, biliary dyskinesia (36 people);
Group V -patients with pathology of the musculoskeletal system (scoliosis, flat feet, hip dysplasia) (21 people);
Group VI -patients with recurrent furunculosis (18);
VII group -patients with chronic granulomatous disease (3 people);
VIII grouphealthy children (12 people).

The survey was additionally carried out a parallel formulation of the NBT-test according to the methods of the claimed method and the prototype method [2]. However, in order to simulate the real working conditions and immunology laboratory, and given the need to obtain reproducible data sufficient for statistical processing, analyses were conducted using four samples of PCT /hereinafter - "NCT-A, NCT-B", "BAT-With", "nst-D/received before the survey is the use of different sources (without explicit violations of the packaging).

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

The results are presented in table 7, 8. In table 7 comparative analysis of the accuracy of FAF in children with various diseases (according to groups) and in healthy children using the inventive method and the prototype method using four samples of CNT (CNT-a And CNT-HCT-C and HCT-E). In table 8, the summary data characterizing the accuracy FAF using the inventive method and the prototype method.

Table 8

Comparison of the accuracy of determining the functional activity of phagocytes /FAF/ using the inventive method and the prototype method (summary results) when using four samples narasinga of tetrazole /CNT, CNT, CNT, CNT-D/ (the determination results FAF confirmed through control of complex examination /KCO/)
The method of determining fatThe cipher used sample PCTCompliance sample nst proposed criterion of suitability for use during the stop NBT-test The number of analyses to determine fat
onlyincluding analyses in which the results of determining fat
were confirmed by the results of KCOwere regarded as erroneous
absed%absed%absed%
123456789
DeclaredPCT-ACC.186100,0186100,000
Nst-Innot ACC.------
Nst-ACC.186100,0186100,000
Nst-Dnot ACC.------
Total372100,0372100,000
Prototype methodPCT--186100,0186100,000
Nst-In-186100,03*1,618398,4
Nst--186100,0186100,000
Nst-D-186100,03*1,618398,4
Total744100,037850,836649,2
* Confirmed the orientation of the irregularities in the system of phagocytosis in the absence of the correlation between relative quantitative values FAF data KCO.

The data are presented in table 7, 8, testify to the following. In the examination of children using the claimed method as a result of preliminary testing four samples of CNT (CNT, CNT, CNT, CNT-D) revealed a discrepancy samples of CNT and CNT-D proposed measure (negative samples nst). In accordance with the claimed method specified negative CNT samples not used in the plantations for the production NBT-test. All results determine PAF claimed process (with the parallel application of positive samples of CNT-a and CNT-fully correlated (as in the direction of the disturbances in the system of phagocytosis and relative /in comparison with the norm/ quantitative values) data KCO in all groups (I-VIII) of the surveyed children (the results are valid in 100% of cases). The formulation of the NBT-test according to the method of the prototype method was carried out with the parallel use of all four samples CNT (CNT-a And CNT-CNT and CNT-D). When using samples of CNT-a and CNT-With (according to the claimed method - positive samples nst) there was complete coincidence of the results of determination FAF with the results of the RUC. When used during production, NBT-test samples of CNT and CNT-D (according to the claimed method - negative samples nst) in 98.4% of children (I-VI, VIII group) were obtained lonesomejohnnie the results of determining fat, which were not confirmed data KCO (either in the direction of the violations or relative /in comparison with the norm/ quantitative values) and did not correspond to data obtained using the prototype method using samples of CNT-a and CNT-C. these lonesomejohnnie the results of determining fat (decrease 95.6-99,3% compared to the norm) was not significantly different between the study groups (ka is sick /I-VI/, and healthy /VIII/ children), and within each group, which included patients with different orientation and intensity of disturbances in the system of phagocytosis set using KCO (group I -38 people with normal fat, 19 people with high fat; group II - 3 people with normal fat, 19 people with high fat; group III - 2 people with normal fat, 15 people with high fat; group IV - 33 persons with normal fat, 3 people with high fat; group VI - 16 persons with low fat, 1 person with normal fat, 1 person with high fat). In 3 children VII group (patients with chronic granulomatous disease) in the formal coincidence of the direction of the disturbances in the system of phagocytosis data KCO quantitative values FAF obtained using samples of CNT and CNT-D, were significantly reduced compared with the values of the percentages obtained using samples of CNT-a and CNT-C (respectively, 0,83±0,45; 0,92±0,36; 0; 0% formazan-positive cells in the smear). Thus, the results of determining fat obtained using the prototype method was reliable only in the case of positive (according to the claimed method) CNT samples. In the case of negative (according to the claimed method) CNT samples the results of determining fat was regarded as wrong almost 100% of cases. So about the time, data analysis table 7, 8 allows to draw the following conclusions. When setting NBT-test in terms of random alternation of CNT samples from different sources (without testing them according to the claimed method) the probability of obtaining false positives determine fat can vary from 0% (in the target group are all used to analyze samples of CNT match the proposed measure) to 100% (in the target group are all used to analyze samples of CNT nesootwetstwujut proposed measure). Conventionally, assuming that in the target group analyses are conducted with the same probability of using positive (according to the claimed method) and negative (according to the claimed method) CNT samples, frequency loosening results will make up 49.2% (table 8).

According to the practical experience of the authors of the invention in real working conditions and immunology laboratory frequency loosening results determine fat obtained when setting NBT-test according to the methods of the prototype method [2] and the method-analogue of [1] in the context of random alternation of CNT samples from different sources, is 25-33%. This suggests that the negative (according to the claimed method) CNT samples are 25-33% of the total number of samples nst coming in immune responsive ness going down the practical laboratory. Thus, the implementation of the claimed method can improve the accuracy FAF at 25-33% due to the exclusion of the possibility of obtaining loosening results, due to the use of CNT samples with the lost ability to recover in formazan.

Sources of information

1. Subic MG, Mednikova VG NBT test in children in normal and purulent bacterial infections // Laboratory work. - 1978. No. 9. - S-517.

2. Immunological methods: TRANS. with it. Ed. Grimes. - M.: Medicine, 1987. - S-338, 378-386, 387-389 (prototype).

3. Nitroblue Tetrazolium. - The Internet.: http: // www. jtbaker.com /msds/ englishhtml / n5100.htm.

4. Handbook of biochemistry: TRANS. from English. / Dawson R., Elliot D., Elliot U., Jones, K. - M.: Mir, 1991. - S.

5. Lilly R. Histopathological technique and practical histochemistry: TRANS. from English. Ed. Vol. - M.: Mir, 1969. - S-134, 304-305.

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

7. Cetlinski S.A., Kalinin NM Immunology for the doctor. - SPb.: LLP Publisher Hippocrates, 1998. - P.113, 143-144.

8. Encyclopedic dictionary of medical terms: 3 tons/ Sec. Ed. CH. - M.: Soviet encyclopedia, 1983. Vol.2. - S.

9. RF patent №2242763, G 01 N 33/53, 20.12.2004, bull. No. 35.

1. The method of determining the functional activity of phagocytes, including taking blood anticoagulant the Ohm, preparation of a mixture for the study of phagocytosis by making the blood sample is pre-prepared with 0.1%solution narasinga of tetrazole, dissolved in 0.9%sodium chloride solution, incubation of the mixture for the study of phagocytosis, preparation of smears from preincubating mixture for the study of phagocytosis and subsequent determination of the number of formazan-positive phagocytes in percent of the total number of phagocytes and assessment of the functional activity of phagocytes by comparing the obtained values with normal values, characterized in that before preparing the mixture for the study of phagocytosis prepare the control mixture, with some pre-prepared solution narasinga of tetrazole volume of at least 0.05 ml mix with the yeast Saccharomyces cerevisiae, maintain control mixture for at least 6 minutes at a temperature of (20±2)°S, and then analyze its color and use the rest of the pre-prepared solution narasinga of tetrazole to prepare the mixture for the study of phagocytosis only if control mix pink colouring.

2. The method according to claim 1, characterized in that mix with the yeast Saccharomyces cerevisiae part of a pre-prepared solution narasinga of tetrazole volume of 0.05-0.1 ml.

3. The method according to claim 1, characterized in that when used for cooking control mix yeast Saccharomyces cerevisiae in a dry form is mixed with solution narasinga of tetrazole at a concentration of 50-250 mg yeast/ml narasinga of tetrazole.

4. The method according to claim 1, characterized in that when used for cooking control mix yeast Saccharomyces cerevisiae as a suspension they are mixed with a solution narasinga of tetrazole concentration (2,5-12,5)·10 yeast cells/ml narasinga of tetrazole.

5. The method according to claim 1, characterized in that the control mixture was incubated for 6-10 minutes

6. The method according to claim 1, characterized in that after incubation of the mixture for the study of phagocytosis additionally centrifuged and prepare smears from obtained after centrifugation of sediment.

7. The method according to claim 6, characterized in that the centrifugation of the mixture for the study of phagocytosis produce for 5 min at 750 rpm



 

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3 ex, 3 tbl

FIELD: immunology.

SUBSTANCE: through a flow microcell of 15-20 mcl volume one should pass the flow of phosphate buffer solution-carrier at the rate of about 30-90 mcl/min which should be supplemented with analyzed serumal sample at the volume of 200 mcl. The flow cell is supplied with a horizontally fixed piezogravimetric immunosensor at fluctuation frequency fm and receptor covering based upon one of the LPC Yersinia enterocolitica of serovars O:3, O:5 or O:6.30. Then it is necessary to register the binding LPC Yersinia enterocolitica of serovars O:3, O:5 or O:6.30 with antibodies according to the decrease of sensor's fluctuation frequency from fm up to f to detect the concentration of antibodies according to calibration graph being directly proportional against ▵f being equal to fm-f. Application of the present innovation enables to decrease the procedure of analysis up to 10 min and, also, carry out a re-usable analysis after regeneration of sorbed LPC at sensitivity being 1.3 mcg/ml and detect concentration of antibodies ranged 10-100 mcg/ml.

EFFECT: higher accuracy and efficiency of detection.

8 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with detecting the concentration of antibodies to neurospecific enolase (NSE) and gliofibrillo-acid protein (GFAP) in blood serum of pregnant women. The development of gestosis should be predicted at the level of anti-NSE-antibodies being above 0.6 mcg/ml or anti-GFAP-antibodies being above 0.9 mcg/ml. The innovation provides high information value and specificity of the method suggested to predict the development of gestosis.

EFFECT: higher efficiency.

3 ex, 1 tbl

FIELD: medicine, ophthalmology.

SUBSTANCE: while inspecting a patients with chorioretinitis it is necessary to fulfill immunogenetic typing of peripheral blood lymphocytes by HLA I class (loci A and B). At detecting antigens HLA-A9 and/or A24 and/or B53 one should predict light flow of chorioretinitis, at detecting antigens HLA-B5(B51) and/or B27 - sever flow and at detecting antigens HLA-B22 and/or B56 - highly severe flow of chorioretinitis. The innovation provides improved quality of diagnostics of chorioretinitis due to predicting the severity degree of the disease flow that enables to prevent irreversible retinal alterations and vascular membrane as it is by prescribing adequate therapy in due time.

EFFECT: higher accuracy of prediction.

3 ex, 2 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: additionally to clinico-anamnestic survey it is necessary to carry out immunoenzymatic assay of a liquor for the content of IL-8, at its level being equal 95 pg/ml and higher one should diagnose hemorrhagic insult, and at IL-8 content being under 95 pg/ml - ischemic insult. The innovation enables to diagnose both ischemic and hemorrhagic insult under inpatient conditions in hospitals during the first days of insult occurred.

EFFECT: higher accuracy of diagnostics.

4 ex, 2 tbl

FIELD: analytical chemistry, immunology.

SUBSTANCE: invention relates to a method for selective determination of nonylphenol that is carried out by using a piezoelectric crystal immunosensor. Sample containing nonylphenol and fixed amount of antibodies raised to its in phosphate buffer (pH = 7.1-7.5) is passed through a flow-type cell comprising mass-sensitive piezoelectric crystal immunosensor with receptor cover based on aminophenol-protein conjugate. Nonylphenol is determined by recording change of the sensor frequency oscillation in interaction of aminophenol-protein conjugate with antibodies to nonylphenol wherein the sensor frequency oscillation change is inversely with the nonylphenol concentration in a sample to be analyzed. Then method involves regeneration of receptor cover for carrying out the following assays by passing 0.02-0.10 mM solution of potassium thiocyanate through a cell. Using the invention provides carrying out multiple selective analysis with the detection limit of nonylphenol 0.8 ng/ml and to determine the concentration of nonylphenol in the range 1-20 ng/ml.

EFFECT: improved assay method.

1 tbl, 13 ex

FIELD: medicine, immunology.

SUBSTANCE: the suggested composition contains cedar oil at the quantity of 5.6-24.4 (weight%) and lipopolysaccharide out of Yersinia enterocolitica at the quantity of 75.6-94.4 (weight%). The application of the present innovation enables to simplify and accelerate the development of covering at the surface of piezoelectric crystal resonator and, also, detect Yersinia enterocolitica antibodies at the range of detection being 1.3 mcg/ml.

EFFECT: higher efficiency.

7 ex, 1 tbl

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

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