Method for studying absorbing and metabolic activity of peripheral blood neutrophils by applying phagocytosis and tetrazole nitro blue

FIELD: medicine.

SUBSTANCE: method involves pouring venous blood treated with heparin into five conic test-tubes in the amount of 0.1 ml. The first three of them contain 0.1 ml of non-colored latex suspension with particle size of 1.5 mcm, the fourth one contains 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue, the fifth one contains .1 ml of latex suspension and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue. The first test-tube is incubated in thermostat for 5 min at37°C, the second one for 30 min, the third one for 1 h, the fourth and the fifth one for 40 min. Smears are prepared from 0.2 ml of incubation mixture on glasses and dried at 37°C, fixed in burner flame, stained with 0.1% aqueous solution of tetrazole nitro blue, repeatedly dried and studied with microscope under immersion with magnification of 90x10. Test results are evaluated from absorption activity in phagocytosis reactions in determining the number of phagocytes, phagocytic number, phagocytic integral index and phagocytosis rate values. Tetrazole nitro blue test response is determined by counting formazan-positive cell number, calculating cytochemical activity index and tetrazole nitro blue test stimulation index.

EFFECT: accelerated test; high accuracy and low cost of examination.

1 dwg, 3 tbl

 

There is a method of determining the absorptive activity of neutrophils peripheral blood, using as the test system of the colored latex particles with a diameter of 3 μm (Medvedev A.N., Chalenko VV Method for studying adsorption phase of phagocytosis // Laboratory business, 1991, No. 2, p.19-20).

There is a method of study of the reactions of bacterial phagocytosis by neutrophils capillary blood (I.V., Nesterov, Slynko LI, Yu M.A., Micenko L.G. detect changes in microbicide system of neutrophils in allergic diseases / guidelines. Krasnodar, 1989, page 11-12).

There is a method of research oxygendependent metabolism of neutrophils peripheral blood, using as test systems laboratory strain of Staphylococcus aureus P-209 (B.H. Park, Fikrig S.M., Smithwich E.M. Infection and nitrobluetetrazolium reduction by neutrophils; a diagnostic aid // Lancet, 1968, vol.11, No. 2, p.532-534).

There is also a way to study the metabolic activity of neutrophils (Wixman M.E. Ter-Minassian, mayansky A.N. Characteristics of opsonic factors for response recovery narasinga of tetrazole by human neutrophils // Bulletin of experimental biology and medicine, 1980, CH, No. 2, s-215).

Any work describes the determination of the adsorption phase of phagocytosis and metabolic activity of neutrophils in the peripheral blood in adca one study, in the literature was not found.

The aim of the invention is to optimize the research time reduction, cost reduction and increase its sensitivity.

This goal is achieved by the fact that heparinized venous blood of the child is poured into five conical tubes 0.1 ml, the first three of which contain 0.1 ml of suspension unpainted latex with a particle size of 1.5 μm, 4-I - 0,1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution narasinga of tetrazole (NBT), 5-I - a 0.1 ml suspension of latex and 0.1 ml of 0.1% aqueous solution of HCT, incubated in a thermostat at 37 t°S: 1st test tube 5 min, 2nd - 30 min, 3-n - 1 h, 4-th and 5-th - 40 min; prepared from 0.2 ml of the incubation mixture smears on slides, dried at 37 t°C, fixed in the burner flame, stained with 1% aqueous solution of methylene blue, re-dried and mikroskopiruyut by immersion in increasing 90×10, and the results of the studies assessed by absorption activity in the reactions of phagocytosis, determining the percentage faguoqitirute cells, phagocytic number, phagocytic integral index and the rate of phagocytosis, and the reaction of NBT-test determine by counting the percentage formazan-positive cells, calculate cytochemical activity indicator and stimulation index NBT-test.

Example 1. Study of the adsorption phase of phagocytosis and metabolic activity of the neutrophils of peripheral blood was conducted in 36 healthy children aged 1 month to 5 years. The first group consisted of 16 children under the age of 1 year (mean age of 4.2±1.1 months), the second group of 20 children aged from 1 year to 5 years (2,9±1.8 years). The dependence of the parameters of the functional activity of neutrophils of peripheral blood from the age of the children is presented in table 1.

Table 1

Indicators of phagocytosis and NBT-test in healthy children
Indexgroup 1(n=16)group 2 (n=20)The reliability of differences
PHAGOCYTOSIS
%F 5 min31,8±6,255,3±7,2P<0,05
FC 5 min2,66±0,782.57 m±0,61 
%F 30 min53,2±7,376,8±5,9P<0,05
FC 30 min2,86±0,323,84±0,48P<0,05
%F 1 hour77,4±3,988,5±5,5P<0,05
FC 1 hour4,47±0,986,69±0,81P<0,05
CFC0,72±0,080,68±0,06 
CFC0,59±0,070,41±0,05P&l; 0,05
SOFIA2,01±0,512,94±0,55 
Spontaneous NBT-test
%PC36,5±4,426,1±3,3P<0,05
LCA0,97±0,090,62±0,03P<0,05
Stimulated NBT-test
%PC50,0±5,844,3±5,6 
LCA1,46±0,111,28±0,17 
INST1,51±0,121,84±0,10P<0,05

Study of the reactions of phagocytosis and NBT-test is performed in parallel, from one portion of the sediment, using identical reagents, dyes, laboratory glassware and equipment.

As test systems using particle unpainted latex (10% polystyrene suspension) with a diameter of 1.5 μm manufactured by Deem, Russia. When setting NBT-test, in addition, use of nicrosini of tetrazole (CNT)40H30CL2N10About6molecular weight 817,65 made in Germany.

The method of phagocytosis. Three conical tubes containing 0.1 ml of a suspension of latex and 0.1 ml of heparinized venous blood, inquiry is in thermostat at 37° With (1-u tube - 5 minutes 2 - 30 minutes, 3-j - 1 hour). Prepare smears of the incubation mixture on the glass, which, after drying, fixation in the flame of the burner, staining with 1% methylene blue mikroskopiruyut with increasing 90×10 under immersion. Take into account data on 100 neutrophils, of which I hope phagocytic activity (%f) -% faguoqitirute neutrophils; phagocytic number (FC) - the average number of particles captured single cell and phagocytic integral index (SOFIA) - the average number of absorbed particles of latex per 100 neutrophils. Calculate the coefficients of phagocytic number (CFC), characterizing the rate of phagocytosis: the ratio FC 5 min to FC 30 min - CFC 1 and the ratio FC 5 min to FC 1 hour - KFC 2.

From the same portion of blood simultaneously with the study of the reactions of phagocytosis is nst-test: 0.1 ml of heparinized blood and 0.1 ml of 0.1% aqueous solution of CNT incubated in a conical test tubes at 37°C for 40 minutes with 0.1 ml of medium 199 spontaneous NBT-test or with 0.1 ml of a suspension of latex - stimulated NBT-test. Similarly, the smear preparation for phagocytosis prepare smears: 0.2 ml of the incubation mixture is applied on glass, dried, fixed, stained with 1% solution of methylene blue, dried and mikroskopiruyut by immersion in increasing 90×10. Assessment of the NBT-test is carried out on the following parameters: % PC - Pro is UNT formazan-positive neutrophils; calculates the index of stimulation of HCT-test (nst IP) is the ratio of % PC in stimulated NBT-test to % PC in spontaneous NBT-test. In order to compute a cytochemical activity indicator (LCA) conduct a visual assessment of the number of granules of deformazione in neutrophils using the principle of Kaplow L.S. by the formula

where a, b, C, d, e - the number of cells, respectively, 0, 1, 2, 3, 4-th class.

Example 2. Child, R. Jan, 7 months, was admitted to the Department of resuscitation and intensive therapy Morozov children's hospital (MDCB) along an ambulance from home with a diagnosis of Acute pneumonia?, respiratory failure II. During the examination the patient was diagnosed with acute polysegmental destructive pneumonia.

On the 3rd, 10th and 30th day of illness the child was the study of the functional activity of neutrophils (table 2). The study was carried out analogously to example 1.

Table 2

The indicators of the functional activity of neutrophils in the peripheral blood of a child R. Jana, 7 months.
Indicators3-day sickness10-day sickness30-day sicknessNormal values
PHAGOCYTOSIS
%the 5 min 2770*3231,8±6,2
FC 5 min3,223,91*2,812,66±0,78
%F 30 min4684*4853,2±7,3
FC 30 min3,266,83*3,742,86±0,32
%F 1 hour60*89*7477,4±3,9
FC 1 hour4,01of 5.92*5,504,47±0,98
CFC0,79*0,83*0,730,72±0,08
CFC0,47*0,75*0,510,59±0,07
SOFIA7,09*4,84*1,422,01±0,51
Spontaneous NBT-test
%PC2860*3436,5±4,4
LCA0,52*0,970,880,97±0,09
Stimulated latex nst-test
%PC34*87*55 50,0±5,8
LCA1,84*2,20*1,551,46±0,11
INST1,21*1,451,621,51±0,12
* - reliability of differences with normal values of P<0,05.

In the study of the dependence of phagocytosis indices on the time of incubation was found that the % f and FC significantly increase over time, reaching maximum values to 1 hour. Upon further incubation indicators phagocytic activity does not change. The drawing shows the dependence of the parameter %f neutrophils from the incubation time in minutes. It is obvious that prolonged incubation, proposed some methods in the study of the absorption phase of phagocytosis using as a test system of the latex particles is not rational. Incubation for 1 hour is sufficient for a reliable evaluation of phagocytic activity of neutrophils, while reducing time spent on research.

It is established that for the simultaneous study of the absorptive phase reactions of phagocytosis and oxygendependent metabolism of neutrophils in the peripheral blood by the method of NBT-test rational use of unpainted latex with a diameter of h is CI 1.5 μm. The use of larger and/or colored latex particles complicates the visual assessment of the stimulated NBT-test principle Kaplow. Smaller latex particles captured by neutrophils in a significant amount (20 to 30 particles per cell), which makes the calculation using an ordinary light microscope.

The influence of the shape of the bottom of the tube is round and tapered. In conical tubes observed higher values as indicators of phagocytosis and NBT-test. Apparently, the increase of the functional activity of neutrophils due to the influence of korotkovolnovogo interaction. This was the criterion for selection of laboratory glassware, as noted in example 1, the study used test tubes conical shape.

In the course of the study according to the absorption capacity and metabolic activity of neutrophils from different stabilizers blood found that Trilon B (disodium salt of EDTA), in contrast to heparin inhibits phagocytosis, reduces oxygendependent metabolism of neutrophils.

Example 3. Child of S. Philip, 5 years, enrolled in the section 8 MDCB with a diagnosis of right-sided polysegmental pneumonia. The study phagocytic and metabolic activity of neutrophils in the peripheral blood was carried out on the 4th d is Ni and on the 22nd day of the disease according to the method described in example 1 (table 3).

Table 3

The indicators of the functional activity of neutrophils in the peripheral blood of a child With. Philip, 5 years
Index4-day sickness22-day sicknessNormal values
PHAGOCYTOSIS
%F 5 min80*6355,3±7,2
FC 5 min2,72,312.57 m±0,61
%F 30 min97*7976,8±5,9
FC 30 min5,43*3,593,84±0,48
%F 1 hour98*8988,5±5,5
FC 1 hour4,84*5,996,69±0,81
CFC0,51*0,640,68±0,06
CFC0,56*0,510,41±0,05
SOFIA5,27*3,312,94±0,55
Spontaneous NBT-test
%PC73*3026,1±3,3
C is A 1,73*0,620,62±0,03
Stimulated latex nst-test
%PC69*5244,3±5,6
LCA1,70*1,331,28±0,17
INST0,94*1,741,84±0,10
* - reliability of differences with normal values of P<0,05.

The present invention not obvious to a person skilled working in the field of clinical immunology and Pediatrics. Determination of the adsorption phase of phagocytosis and metabolic activity of neutrophils in the peripheral blood in the order of one study have not previously held, the very posing of such a question seemed absurd. The novelty of the invention lies in the possibility of conducting an investigation of two methods using one portion of the sediment (blood), identical reagents and equipment. This allows you to optimize the method, makes it available in infants and newborns and improve their comprehension. Combining two different techniques require long research and hard work on the selection of optimal conditions, allowing at the same time, saving time, R the assets and lesser invasiveness, to provide high information content studies.

The claimed method has important social and economic value. It allows you to save time and reagents, does not require expensive equipment, creates a previously unknown examination of children's groups to identify unexpected results, which can be the basis for the diagnosis of various pathological conditions. Application equipment standard clinical laboratory not only leads to reduction of material costs, but also opens the prospect of the introduction of the proposed research to study the parameters of non-specific immunity in an outpatient setting. The reduction of labor costs, saving time creates a potential opportunity to conduct research as a screening test in the examination of different groups of children, which is unknown to the specialists working in the field of clinical immunology and Pediatrics.

Method of study the absorption and metabolic activity of neutrophils in peripheral blood in children methods of phagocytosis and NBT-test, wherein heparinized venous blood poured in five conical tubes 0.1 ml, the first three of which contain 0.1 ml of suspension unpainted latex with a particle size of 1.5 μm, 4-I - 0,1 ml of medium 199 and 0.1 ml of 0.1% vodno the solution narasinga of tetrazole (PCT), 5-I - a 0.1 ml suspension of latex and 0.1 ml of 0.1% aqueous solution of HCT, incubated in a thermostat at 37 t°S: 1st test tube 5 min, 2nd - 30 min, 3-n - 1 h, 4-th and 5-th - 40 min; prepared from 0.2 ml of the incubation mixture smears on slides, dried at 37 t°C, fixed in the burner flame, stained with 1% aqueous solution of methylene blue, re-dried and mikroskopiruyut the immersion with increasing h, and the results of the studies assessed by absorption activity in the reactions of phagocytosis, determining the percentage faguoqitirute cells, phagocytic number, phagocytic integral index and the rate of phagocytosis, and the reaction of NBT-test determine by counting the percentage formazan-positive cells, calculate cytochemical activity indicator and stimulation index NBT-test.



 

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FIELD: medicine, analytical biochemistry.

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EFFECT: higher accuracy of evaluation.

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EFFECT: higher accuracy of detection.

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EFFECT: high accuracy of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves studying biological material by applying infrared spectroscopy techniques. The obtained data are processed and diagnosis is set. Blood serum is used as the biological material. The study is carried out by preparing dried blood serum sample as suspension in Vaseline oil and doing the infrared spectroscopy analysis in the bandwidth of 120-1000 cm-1 and determining absorption strip peak heights having maximum at 1170; 1165; 1160; 1150; 1140; 1060; 1050; 1040; 1025 and then calculating the following ratio values like peak height with maximum at 1160 cm-1 to 1140 cm-1; 1165 cm-1 to 1150 cm-1; 1040 cm-1 to 1025 cm-1. The obtained distribution of this group is projected to frontal plane for calculating two-dimensional coordinates and comparing to flat reference diagnostic images of prostate pathologies and to a normal reference diagnostic image represented as flat polygons which boundaries are given by the following values. The norm is represented by X(-1.15;-0.9;0.45;0.0;-0.65) and Y(0.99;4.2;0.9;0.46), respectively. Pathology by X(-1.15;-1.15;0.35;0.0;0.65) and Y(0.99;-0.03; 0.48;0.09;0.46). The norm and pathology are differentiated. Additional mathematical processing is carried out on infrared spectra of blood serum samples of patients belonging to pathology image according to parameter values. First of all, three-dimensional distribution is calculated as peak having maximum at 1160 cm-1 to one having maximum at 1150 cm-1; 1170; 1160 cm-1; 1160 cm-1 to 1025 cm-1. It is projected then to frontal plane and compared to diagnosis images of prostate adenoma and images of prostate carcinoma. The second group relationships the following values are used: oncological cases - X(0.28;0.77;1.24;0.96), Y(0.75;0.46;-0.13;-0.02); adenoma - X(0.28;1.24;2.21;1.24;0.77), Y(0.75;1.24;-0.12;-0.13;0.46). Differential diagnosis of pathologies is set by interpreting point position within particular pathology image.

EFFECT: high accuracy of differential diagnosis.

FIELD: medicine.

SUBSTANCE: method involves determining mean cytochemical coefficient of lipid accumulation in peripheral blood leukocytes in conditional units before beginning therapy application (MCC1) and in 2-3 or 5-6, or 10-12, or 20-24 months of therapy application (MCC2). Therapy effectiveness coefficient is calculated in conditional units from formula K= MCC2/MCC1. The value being equal to or greater than 1, leprosy therapy is predicted to be effective.

EFFECT: simplified prognosis method.

1 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves determining infrared radiation absorption coefficient in blood plasma in bandwidth of 1543-1396 cm-1. The infrared radiation absorption coefficient is determined in %. The value being equal to 29.7±1.1%, catarrhal cholecystitis is diagnosed. The value being 26.4±1.4%, phlegmonous cholecystitis is diagnosed. The value being 21.2±1.8%, gangrenous cholecystitis is diagnosed. The value being equal to 18.6±0.5%, gangrenous perforated cholecystitis case is diagnosed. The value in norm is equal to 32.4±0.8%.

EFFECT: high accuracy and specificity of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves pouring venous blood treated with heparin into five conic test-tubes in the amount of 0.1 ml. The first three of them contain 0.1 ml of non-colored latex suspension with particle size of 1.5 mcm, the fourth one contains 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue, the fifth one contains .1 ml of latex suspension and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue. The first test-tube is incubated in thermostat for 5 min at37°C, the second one for 30 min, the third one for 1 h, the fourth and the fifth one for 40 min. Smears are prepared from 0.2 ml of incubation mixture on glasses and dried at 37°C, fixed in burner flame, stained with 0.1% aqueous solution of tetrazole nitro blue, repeatedly dried and studied with microscope under immersion with magnification of 90x10. Test results are evaluated from absorption activity in phagocytosis reactions in determining the number of phagocytes, phagocytic number, phagocytic integral index and phagocytosis rate values. Tetrazole nitro blue test response is determined by counting formazan-positive cell number, calculating cytochemical activity index and tetrazole nitro blue test stimulation index.

EFFECT: accelerated test; high accuracy and low cost of examination.

1 dwg, 3 tbl

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