RU2249215C2 - Method for studying absorbing and metabolic activity of peripheral blood neutrophils by applying phagocytosis and tetrazole nitro blue - Google Patents

Abstract

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

Description

A known method for determining the absorption activity of peripheral blood neutrophils using as a test system colored particles of latex with a diameter of 3 μm (Medvedev A.N., Chalenko V.V. Method for the study of the absorption phase of phagocytosis // Laboratory work, 1991, No. 2, p. 19-20).

A known method of studying the reactions of bacterial phagocytosis with neutrophils of capillary blood (Nesterova I.V., Slynko L.I., Svetlichnaya M.A., Maychenko L.G. Diagnosis of changes in the microbicidal system of neutrophilic granulocytes in allergic diseases / Methodical recommendations. Krasnodar, 1989 , pp. 11-12).

A known method for studying the oxygen-dependent metabolism of peripheral blood neutrophils using the laboratory strain Staphylococcus aureus P-209 (Park BH, Fikrig SM, Smithwich EM Infection and nitrobluetetrazolium reduction by neutrophils; a diagnostic aid // Lancet, 1968, vol. 11 No. 2, p. 532-534).

There is also a method for studying the metabolic activity of neutrophils (Vicksman M.E., Mayansky A.N. Characterization of opsonic factors by the reaction of nitro blue tetrazolium reduction by human neutrophils // Bulletin of Experimental Biology and Medicine, 1980, vol. 89, No. 2, p.214- 215).

There are no works that describe the determination of the absorption phase of phagocytosis and the metabolic activity of peripheral blood neutrophils in the order of one study.

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

This goal is achieved by the fact that heparinized venous blood of a child is poured into five 0.1 ml conical tubes, the first three of which contain 0.1 ml of a suspension of unpainted latex with a particle size of 1.5 μm, the fourth - 0.1 ml medium 199 and 0.1 ml of a 0.1% aqueous solution of nitro-blue tetrazolium (HCT), 5th - 0.1 ml of a suspension of latex and 0.1 ml of a 0.1% aqueous solution of HCT, incubated in a thermostat at t 37 ° C : 1st tube - 5 min, 2nd - 30 min, 3rd - 1 hour, 4th and 5th - 40 min; prepared from 0.2 ml of the incubation mixture, smears on glasses, dried at t 37 ° C, fixed in the flame of the burner, stained with a 1% aqueous solution of methylene blue, re-dried and microscopic under immersion at a magnification of 90 × 10, and the results of the study are evaluated by absorption activity in phagocytosis reactions, determining the percentage of phagocytic cells, phagocytic number, phagocytic integral index and phagocytosis rate indicators, and the HCT test reaction is determined by calculating the percentage of formazan-positive cells, calculated cytochemical activity indicator and stimulation index of the HCT test.

Example 1. The study of the absorption phase of phagocytosis and metabolic activity of peripheral blood neutrophils 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 (average age - 4.2 ± 1.1 months), the second group - 20 children aged 1 year to 5 years (2.9 ± 1.8 years). The dependence of the functional activity of peripheral blood neutrophils on the age of children is presented in table 1.

Table 1
Indicators of phagocytosis and HCT test in healthy children Indicator 1 group (n = 16) 2 group (n = 20) Confidence Differences PHAGOCYTOSIS % F 5 min 31.8 ± 6.2 55.3 ± 7.2 P <0.05 Ff 5 min 2.66 ± 0.78 2.57 ± 0.61 % F 30 min 53.2 ± 7.3 76.8 ± 5.9 P <0.05 PS 30 min 2.86 ± 0.32 3.84 ± 0.48 P <0.05 % F 1 hour 77.4 ± 3.9 88.5 ± 5.5 P <0.05 1 hour 4.47 ± 0.98 6.69 ± 0.81 P <0.05 KFCH1 0.72 ± 0.08 0.68 ± 0.06 KFCH2 0.59 ± 0.07 0.41 ± 0.05 P <0.05 FII 2.01 ± 0.51 2.94 ± 0.55 Spontaneous HCT Test %PC 36.5 ± 4.4 26.1 ± 3.3 P <0.05 CPA 0.97 ± 0.09 0.62 ± 0.03 P <0.05 Stimulated HCT Test %PC 50.0 ± 5.8 44.3 ± 5.6 CPA 1.46 ± 0.11 1.28 ± 0.17 ISNST 1.51 ± 0.12 1.84 ± 0.10 P <0.05

The study of phagocytosis and HCT test reactions is carried out in parallel, from one portion of the biosubstrate, using identical reagents, dyes, laboratory glassware and equipment.

Unpainted latex particles (10% polystyrene suspension) with a diameter of 1.5 μm manufactured by DIAeM, Russia, are used as a test system. When staging the HCT test, in addition, nitrosine tetrosolium (HCT) - C ₄₀ H ₃₀ Cl ₂ N ₁₀ O ₆ with a molecular weight of 817.65 manufactured in Germany is used.

The technique of phagocytosis. Three conical tubes containing 0.1 ml of latex suspension and 0.1 ml of heparinized venous blood are incubated in an incubator at 37 ° C (1st tube - 5 minutes, 2nd tube - 30 minutes, 3rd tube - 1 hour) . Prepare smears of the incubation mixture on glass, which, after drying, fixing in the flame of the burner, staining with 1% methylene blue, are microscopically magnified at 90 × 10 under immersion. Data on 100 neutrophils are taken into account, from which phagocytic activity (% F) is calculated - the percentage of phagocytic neutrophils; phagocytic number (PS) is the average number of particles captured by one cell, and phagocytic integral index (PHI) is the average number of latex particles absorbed per 100 neutrophils. The phagocytic number coefficients (KPF), which characterize the rate of phagocytosis, are calculated: the ratio of the 5-min PS to the 30-PS PS - 1 and the 5-minute PS-5 ratio to 1-PSK 2.

From the same portion of blood, simultaneously with the study of phagocytosis reactions, an HCT test is performed: 0.1 ml of heparinized blood and 0.1 ml of a 0.1% aqueous solution of HCT are incubated in conical tubes at 37 ° C for 40 minutes with 0.1 ml Wednesday 199 - spontaneous HCT test or with 0.1 ml latex suspension - stimulated HCT test. Similar to the preparation of a smear for phagocytosis, smears are prepared: 0.2 ml of the incubation mixture is applied to glass, dried, fixed, stained with 1% methylene blue solution, dried and microscopic under immersion at a magnification of 90 × 10. Assessment of the NST test is carried out according to the following indicators:% PC - percentage of formazan-positive neutrophils; the stimulation index of the HCT test (IC HCT) is calculated - the ratio of% PC in the stimulated HCT test to% PC in the spontaneous HCT test. In order to calculate the cytochemical activity index (CPA), a visual assessment of the number of diformazan granules in neutrophils is carried out using the Kaplow L.S. principle. according to the formula

where a, b, c, d, e are the number of cells, respectively, 0, 1, 2, 3, 4 degrees.

Example 2. A child R. Yana, 7 months old, was admitted to the intensive care unit of the Morozov Children's Clinical Hospital (MDKB) along with an ambulance from home with a diagnosis of Acute pneumonia ?, respiratory failure of the II degree. An examination in a hospital revealed acute polysegmental destructive pneumonia.

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

table 2
Indicators of the functional activity of neutrophils in the peripheral blood of a child R. Yana, 7 months. Indicators 3rd day of illness 10th day of illness 30th day of illness Normal values PHAGOCYTOSIS % F 5 min 27 70 * 32 31.8 ± 6.2 Ff 5 min 3.22 3.91 * 2.81 2.66 ± 0.78 % F 30 min 46 84 * 48 53.2 ± 7.3 PS 30 min 3.26 6.83 * 3.74 2.86 ± 0.32 % F 1 hour 60 * 89 * 74 77.4 ± 3.9 1 hour 4.01 5.92 * 5.50 4.47 ± 0.98 KFCH1 0.79 * 0.83 * 0.73 0.72 ± 0.08 KFCH2 0.47 * 0.75 * 0.51 0.59 ± 0.07 FII 7.09 * 4.84 * 1.42 2.01 ± 0.51 Spontaneous HCT Test %PC 28 60 * 34 36.5 ± 4.4 CPA 0.52 * 0.97 0.88 0.97 ± 0.09 Latex Stimulated HCT Test %PC 34 * 87 * 55 50.0 ± 5.8 CPA 1.84 * 2.20 * 1.55 1.46 ± 0.11 ISNST 1.21 * 1.45 1,62 1.51 ± 0.12 * - significance of differences with normal values of P <0.05.

In the study of the dependence of phagocytosis indices on the incubation time, it was found that% F and PS significantly increase in time, reaching maximum values by 1 hour. With further incubation, phagocytic activity indicators do not change. The drawing shows the dependence of the% F neutrophils from the incubation time in minutes. Obviously, the long-term incubation, proposed in some methods, when studying the absorption phase of phagocytosis using latex particles as a test system, is not rational. Incubation for 1 hour is sufficient for a reliable assessment of the phagocytic activity of neutrophils, while reducing the time spent on the study.

It was established that for the simultaneous study of the absorption phase in the reactions of phagocytosis and oxygen-dependent metabolism of peripheral blood neutrophils by the HCT test, it is rational to use unpainted latex with a particle diameter of 1.5 μm. The use of larger and / or stained latex particles makes it difficult to visually evaluate the Kaplow stimulated HCT test. Smaller particles of latex are captured by neutrophils in a significant amount (20-30 particles per cell), which makes it difficult to count using a conventional light microscope.

The influence of the shape of the bottom of the tube - round and conical. In conical tubes, higher values of both phagocytosis and HCT test values are observed. Apparently, the increase in the functional activity of neutrophils is due to the influence of short-distance interaction. This served as a criterion for the selection of laboratory glassware, as was noted in Example 1, conical tubes are used in the study.

In the course of the study of the dependence of the absorption capacity and metabolic activity of neutrophils on various blood stabilizers, it was found that Trilon B (disodium EDTA), in contrast to heparin, suppresses phagocytosis and reduces the oxygen-dependent metabolism of neutrophils.

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

Table 3
Indicators of the functional activity of neutrophils in the peripheral blood of a child S. Philip, 5 years old Indicator 4th day of illness 22nd day of illness Normal values PHAGOCYTOSIS % F 5 min 80 * 63 55.3 ± 7.2 Ff 5 min 2.7 2,31 2.57 ± 0.61 % F 30 min 97 * 79 76.8 ± 5.9 PS 30 min 5.43 * 3,59 3.84 ± 0.48 % F 1 hour 98 * 89 88.5 ± 5.5 1 hour 4.84 * 5.99 6.69 ± 0.81 KFCH1 0.51 * 0.64 0.68 ± 0.06 KFCH2 0.56 * 0.51 0.41 ± 0.05 FII 5.27 * 3.31 2.94 ± 0.55 Spontaneous and HCT test %PC 73 * thirty 26.1 ± 3.3 CPA 1.73 * 0.62 0.62 ± 0.03 Latex Stimulated HCT Test %PC 69 * 52 44.3 ± 5.6 CPA 1.70 * 1.33 1.28 ± 0.17 ISNST 0.94 * 1.74 1.84 ± 0.10 * - significance of differences with normal values of P <0.05.

The present invention is not obvious to a person skilled in the field of clinical immunology and pediatrics. The determination of the absorption phase of phagocytosis and the metabolic activity of peripheral blood neutrophils in the order of one study has not been previously carried out; the very formulation of this question seemed absurd. The novelty of the invention lies in the possibility of conducting in the order of one study of two methods using one portion of biosubstrate (blood), identical reagents and equipment. This allows you to optimize the method, makes it available in young children and newborns, increases its information content. The combination of two different methods required lengthy research and painstaking work on the selection of optimal conditions, which at the same time save time, reagents and less invasiveness, to ensure high information content of the study.

The claimed method has important social and economic importance. It allows you to save time and reagents, does not require expensive equipment, creates a previously unknown opportunity to examine children's groups with the identification of unexpected results that can form the basis for the diagnosis of various pathological conditions. The use of standard clinical laboratory equipment leads not only to lower material costs, but also opens up the prospect of introducing the proposed study to study the parameters of non-specific immunity on an outpatient basis. Reducing labor costs, saving time creates the potential for conducting research as a screening test when examining various children's groups, which is unknown to specialists working in the field of clinical immunology and pediatrics.

Claims (1)

A method for studying the absorption and metabolic activity of peripheral blood neutrophils in children by phagocytosis and HCT test, characterized in that heparinized venous blood is poured into five 0.1 ml conical tubes, the first three of which contain 0.1 ml of a suspension of unpainted latex with particle size 1.5 μm, 4th — 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of nitros blue tetrazolium (HCT), 5th — 0.1 ml of latex suspension and 0.1 ml 0 , 1% aqueous HCT solution, incubated in a thermostat at t 37 ° C: 1st test tube - 5 minutes, 2nd test - 30 minutes, 3rd test - 1 h, 4th test and 5 th - 40 min; they are prepared from 0.2 ml of the incubation mixture, smears on glasses, dried at t 37 ° C, fixed in the flame of the burner, stained with 1% aqueous solution of methylene blue, re-dried and microscopic under immersion at a magnification of 90x10, and the results of the study are evaluated by absorption activity in phagocytosis reactions, determining the percentage of phagocytic cells, phagocytic number, phagocytic integral index and phagocytosis rate indicators, and the HCT test reaction is determined by calculating the percentage of formazan-positive cells, calculated cytochemical activity indicator and stimulation index of the HCT test.