RU2192638C2 - Method for estimating the differentiation of t-, b-, nk-lymphocytes in blood smears of animals and poultry - Google Patents

Abstract

FIELD: veterinary medicine. SUBSTANCE: the innovation provides the opportunity for simultaneous estimation of peculiarly important cells participating in development of nonspecific body resistance, estimation of nonspecific esterase activity and higher economic efficiency. To prepare buffer mixture one should use mono-potassium phosphate (KH₂PO₄) and disubstituted sodium phosphate (Na₂HPO₄), then incubation mixture is supplemented with a buffer mixture, incubation of smears is conducted in the darkness for 2 h, after drying they are additionally dyed with azure blue-II dye. The method provides better results of immunological trials due to the process of material obtaining and processing. EFFECT: higher efficiency of estimation. 1 cl, 2 dwg

Description

 The invention relates to the field of veterinary medicine, in particular immunology of agricultural animals and birds.

 A solution is known where the determination of lymphocyte populations in the blood of cattle and sheep is carried out by immunochemical analysis of their surface in the reactions of rosette formation using red blood cell markers (see "Immunological research methods in animal husbandry", Methodological recommendations, Lviv, 1987, p. 36.).

 Immunological studies are also known (see Z. Loyda, R. Gossray, T. Schbler. Histochemistry of enzymes. Laboratory methods. Translated from English by Ph.D. IB Bukhvalova, Ph.D. O.V. .Kopieva, under the editorship of Professor N.T. Reichlina publishing house Mir Moscow 1982. p. 103), including the preparation of a biological substrate, its treatment with a buffer-incubation mixture containing phosphate buffer, α-naphthylacetate with acetone and a neutral strong blue dye, incubation of the biological substrate, followed by drying and microscopy.

 A disadvantage of the known technical solutions is the difficulty in obtaining a buffer mixture of a certain pH, the inability to stain the nuclei of lymphocytes, because B cells are determined by the color of the nucleus.

 The technical solution to the problem is to increase the effectiveness of immunological studies by eliminating the loss of leukocytes in the process of obtaining and processing the material, providing the possibility of simultaneously identifying particularly important cells involved in the formation of nonspecific resistance of the body, and determining economic efficiency.

The objective is achieved in that in the method for determining the differentiation of T-, B-, NK-lymphocytes in blood smears of animals and birds, which includes preparing a biological substrate, treating it with a buffer incubation mixture containing phosphate buffer, α-naphthyl acetate with acetone and a neutral dye solid blue, incubation of the biological substrate, staining, followed by drying and microscopy, blood smears of animals and birds are used as the biological substrate, monosubstituted are used to prepare the buffer mixture first phosphoric acid potassium (KH ₂ PO ₄ ) and disubstituted phosphoric acid sodium (Na ₂ HRO ₄ ). Then, 1.2 - 1.3 ml of the buffer mixture is added to the incubation mixture to provide a pH of 8.0 - 8.2 medium. In this case, the smears are incubated in the dark for 2 hours, and after drying, the smears are additionally stained with Azur-II paint in an aqueous 0.25% solution for 0.5 - 1 min, washed and dried. Fixation of smears is carried out in pairs of 40% formalin.

The novelty of the claimed proposal is due to the fact that salts with buffering properties are used to prepare the buffer mixture to obtain a certain pH of the medium, in addition, due to the additional staining of the cell nuclei in blood smears, B-lymphocytes can be determined, in addition, the claimed proposal is express a method for determining nonspecific esterase in blood cells: T, NK-lymphocytes. At the same time, in one preparation, B cells can be determined by the color of the nucleus. Application of the method is based on the interaction of nonspecific esterase with α-naphthyl acetate. Esterase promotes the hydrolysis of α-naphthyl acetate to form 1-naphthol and acetic acid, which creates a slightly acidic environment. To maintain the pH in the range of 8.0–8.2, the buffer mixture proposed by us consisting of KH ₂ PO ₄ and Na ₂ HRO ₄ was introduced into the system, which influenced the activity of the esterase enzyme. Then 1-naphthol interacts with a dye - neutral strong blue B to form a complex of azo dye. The resulting complex (azo dye) stains esterase contained within T and NK lymphopites.

Nonspecific Esterase
The primary reaction (enzymatic digestion reaction) of the hydrolysis of naphthyl acetate with the participation of the esterase enzyme (see Scheme 1 at the end of the description).

 2) Secondary reaction (coupling reaction), i.e. interaction of 1-naphthol with a neutral strong blue B (see Scheme 2).

An example of a specific implementation of the method for determining the differentiation of T-, B-, NK-lymphocytes in blood smears of animals and birds
Blood collection and smear preparation
Blood was taken for examination from farm animals, for example from cows, from the jugular vein, from a bird from the wing vein. A smear was prepared from blood. For this, a drop of blood was applied to the edge of dry fat-free glass, which was held between the thumb and middle finger of the left hand. In front of the droplets, at the angle of 45 ^{°, the} polished edge of the coverslip was brought in so that the angle formed between the glasses was uniformly filled with blood. With the movement of the right hand, a drop was distributed with a thin layer on the surface of a glass slide without gaps in the form of a uniform strip not extending beyond its edges. Smears are dried in air and fixed in pairs of 40% formalin solution for 5 minutes. To do this, about 150 ml of formalin was poured onto the bottom of the desiccator, left under a closed lid for 30 minutes to sublimate formalin vapors. Then, the lid was carefully removed and smears were placed on the desiccator grate and closed with the lid. Thus, smears are fixed.

Buffer Preparation
At the beginning, the pH-150 ionomer was calibrated. The buffer mixture consisted of: monosubstituted phosphoric acid potassium (KH ₂ PO ₄ with a molecular weight of 136.1 g / mol), from which the A solution was prepared; disubstituted phosphoric acid sodium (Na ₂ НСО ₄ with a molecular weight of 177.9 g / mol), from which the solution was prepared - B;
Preparation of Solution A
To prepare 25 ml of solution A, 0.34 g or (340 mg) of KH ₂ PO _{4 was} taken and dissolved in a small amount of distilled water, then brought to the mark in a 25 ml volumetric flask.

Preparation of solution B
8.9 g of Na ₂ HRO _{4 was} taken and dissolved in a small amount of distilled water, then in a 500 ml volumetric flask was brought to the mark.

 Then the flasks with solutions A and B were left for a day in a dark place for better diffusion. The next day after further stirring, 25 ml of solution A and 475 ml of solution B were mixed, mixed thoroughly (preferably left for a day) and then the pH of the buffer solution was measured. It is very important to obtain a pH in the range of 8.0 to 8.2.

Preparation of the incubation mixture
Proper preparation of the incubation mixture is of no small importance for the further staining of smears. Prepare the incubation mixture preferably in a darkened room. To prepare it, prepare solution 1 and 2.

Preparation of solution 1
α-Naphthyl acetate is taken 2.5 mg. For dissolution, 1.25 ml of acetone was taken, diluted with the same amount of distilled water. Naphthyl acetate was dissolved, gradually adding acetone in small portions, stirring. Then, 6.25 ml of distilled water was also gradually added.

Solution 2 preparation
Take 6.25 mg of neutral strong blue dye and dissolve it in 6.25 ml of distilled water, mixing them gradually. To maintain the pH, add 1.25 ml of a buffer solution with a pH of 8.0-8.2.

 The resulting solutions are mixed, filtered in the dark, preferably through a glass filter.

 Smear incubation.

 Smear incubation is carried out exclusively in the dark for 2 hours. On formalin-fixed smears, an incubation mixture is applied evenly. Periodically it is necessary to add the mixture, as liquid evaporation occurs, which includes volatiles (acetone, naphthyl acetate). After incubation, the smears are washed with distilled water and dried in air.

 To differentiate lymphocytes from other blood cells, we further stain the nuclei of the cells with Azur-II paint with an aqueous 0.25% solution for 30 seconds to 1 minute. After painting, the paint is poured, washed with distilled water, the smears are dried in air.

Smear microscopy
Microscopy is performed using an emersion system at magnification (90 x 15).

The results of the data obtained by smear smears:
T-lymphocytes contain one go several granules of dark color, located to the periphery of the cell.

 NK cells are stained diffusely gray.

 B-lymphocytes have colored nuclei, cells do not contain granules (Fig. 1, 2).

Claims (2)

1. A method for determining the differentiation of T-, B-, NK-lymphocytes in blood smears of animals and birds, including the preparation of a biological substrate, processing it with a buffer-incubation mixture containing phosphate buffer, α-naphthyl acetate with acetone and a neutral strong blue dye, biological incubation substrate, followed by drying and microcopy, characterized in that blood smears of animals and birds are used as a biological substrate, and monosubstituted phosphoric acid potassium is used to prepare the buffer mixture ( H ₂ PO ₄₎ and phosphoric acid dibasic sodium (Na ₂ HP0 _4), then the incubation mixture was added 1.2-1.3 ml of a buffer mixture to provide a pH 8.0-8.2, wherein the incubation is performed in the smear darkness for 2 hours, and after drying, smears are additionally stained with AZUR-II paint with an aqueous 0.25% solution for 0.5-1 minutes, washed and dried.  2. The method according to p. 1, characterized in that the fixation of the smears is carried out in pairs of 40% formalin and when preparing the incubation mixture, α-naphthyl acetate is taken in an amount of 2.4-2.6 mg, diluted with an aqueous-acetone mixture prepared in a ratio of 1 : 1 and gradually added to α-naphthyl acetate, then 6.2-6.3 mg of a neutral strong blue dye is added.

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