The visualization of blood cells of birds on a single smear

 

The invention relates to the field of veterinary medicine. A sample of unstabilized blood electron microscope paint dye diamond crazylove blue for 20-40 minutes at a temperature of 18-20oC. In this preliminary mixing of blood with the dye to produce laminar flow in a confined space for 30 s at the same temperature. After cooking and drying of smears made a commit in the solution-the latch on Leishman for 3 min, and then Domracheva diluted buffer solution with a pH of 6.8 to 7.2, consisting of 0,95% sodium phosphate disubstituted and 0,907% potassium phosphate one-deputizing dye-release on Leishman within 10 minutes Smear was washed with the same buffer solution. The proposed method allows one smear simultaneously visualize erythrocytes, reticulocytes, basophils, eosinophils, pseudosinella, lymphocytes, monocytes and platelets. 3 C.p. f-crystals, 4 PL.

The visualization of blood cells of birds relates to veterinary medicine and can be used for bulk studies in laboratory diagnosis.

There are various ways to determine the number of formed elements of blood on the painted samples of fresh chromosomal when using the microscope to see cells, leukocytes, platelets. However, the time of staining, the samples for the detection of erythrocytes and leukocytes varies considerably from 20 minutes to 1 hour (1), (due to the fact that each preparation is obtained the dye with a different title), and to identify platelets duration staining is increased to 1.5-3 hours in Addition, this method does not allow to detect reticulocytes.

Coloring May-Grunwald is carried out without prior fixation (paint dissolved in methyl alcohol), but on the background of well-stained cytoplasm of erythrocytes kernel procrasinate in a light blue color, which hinders karyometry.

Painting on Papenheim combines color and May-Grünwald and counterstaining by Romanovsky, which increases the time of the survey, and as a result well-stained basophilic structure of nuclei and specific grain of leukocytes, which allows to use this method for painting punctata bone marrow, you cannot clearly be differentiated cell population in the peripheral blood.

Coloring Azur-eosin-based dyeing dye Romanovsky, prepared in methyl alcohol, and the fixation and staining are at the same time. In eosinophils - acidified with azure-eosin, i.e. actually conduct two studies.

Staining dye on Leishman involves the use of non-fixed smears, as the paint is prepared on the latch methyl alcohol. This method is economical paint smear takes 13 min (first 3 min swab placed in undiluted dye-fixative, in the next 10 min Dobrescu produce the diluted dye) (2). The disadvantage of this method is the use of distilled water to prepare a diluted dye and wash strokes: if the pH of the environment leads to poor strokes, which reduces the reliability of the results.

A common disadvantage of the above methods is the inability to identify reticulocytes.

In Hematology for counting reticulocytes as dye use: brilliantissima, methylene blue, azure I, Azur II.

The color of the blood smear birds methylene blue, azure I does not differentiate between red blood cells and platelets (due to the same staining nuclei and weak staining of intracellular cytoplasmic structures).

Coloring dye, brilliantissima on the glass in a humid chamber does not identify elementov blood we have chosen the way electron microscope coloring-Azur II (method Alekseev), recommended for the simultaneous detection of reticulocytes and platelets: first acquire a bluish color with typical setdataset, the second - purple-pink (3). However, the staining in melanura reduces the reliability of the results due to mechanical damage to the blood cells of birds with stirring. This method does not allow to visualize the cells of the lymphocytic series, which requires to calculate each cell population in the blood of conducting independent research.

The task of the invention is to provide a method of visualization of blood cells, allowing a single smear counted leucoformula, reticulocytes, platelets, and erythrocytopenia and karyometry, which will increase the reliability of the results and reduce the time of the study.

To achieve the mentioned technical result is offered in the way Alekseeva, including the mixing of blood with azure II dye (by three times the movement of the mixture from melanura on a watch glass and back into melanger), - carrying out dyeing in melanger for 0.5 - 2 hours, stirring the mixture in melanura within 2 minutes, the drying is done on slides smear - Foy water, - counterstaining Romanovsky-Giemsa for 30 to 45 minutes to make the following changes: - to make coloring unstabilized blood, instead Azur II to use 1% solution of the dye brilliantissima,
- stir the blood with dye (gently) for 30 s in a confined space, such as in a test tube at room temperature (18-20oC),
- to paint within 20-40 minutes,
- smears prepared and dried in air, fixed for 3 minutes in a solution-the latch on Leishman;
to zakrashivaet for 10 minutes, dilute the dye solution on Leishman prepared in a buffer solution at a ratio of 1:1, and the buffer is prepared from 0,95% sodium phosphate disubstituted and 0,907% potassium phosphate one-deputizing (pH 6.8 to 7.2);
instead of distilled water to rinse the swab in the same buffer to obtain high-quality smears important maintaining the same pH conditions during coating and rinsing stroke).

Microscope be under the immersion.

The proposed method eliminates the artifacts and damage cells. Much better coverage of the majority of cellular structures reduces the duration of studies due to the fact that one of musicista reticulocytes, platelets, as well as erythrocytopenia and karyometry.

Distinctive features of the claimed method is that the staining unstabilized blood produce a 1% solution of brilliantissima for 20-40 minutes at a temperature of 18-20oWith, and to ensure the safety of blood cells merging and laminar mixing of blood with the dye is carried out in a confined space, such as in a test tube for 30 s at the same temperature; cooked and dried smear is fixed in a concentrated solution for Leishman for 3 min; Domracheva it in dilute solution by Leishman prepared in buffer solution for 10 min, and washed in the same buffer solution.

Laminar mixing of the dye and the blood makes it possible to retain cells in the undeformed state and provides coverage of cellular structures not only erythroid, and lymphocytic series.

Electron microscope coloring allows you to save and to identify the intracellular elements containing ribonucleoprotein, while committing to smear staining leads to their destruction.

Subsequent fixation electron microscope colored smears in the solution-dye on Leishman provide the staining on Leishman in dilute solution, prepared in buffer solution, and rinsing in the same buffer solution to allow you to maintain a constant pH and microscopy under immersion to get a true picture of blood cells due to differential staining of each type of cells and intracellular structures. The proposed method allows to visualize simultaneously on a single smear all formed blood elements, and
- red blood cells are elongated ellipsoids with sharply OxyFile cytoplasm rich in hemoglobin, the core of an intense blue-violet colour with dense chromatin structure;
- reticulocytes - elongated ellipsoids with clearly allocated grained thread-substance in the form of blue veins on pink (exefilename) background and core from light blue to violet color;
- basophils - dark purple cells are rounded with a poorly visible nuclei due to graininess, which is superimposed on the nuclear and cytoplasmic patterns;
- eosinophils - cells rounded shape with an eccentric located light purple core and light blue cytoplasm with round grain from dark red to light pink color;
- pseudosingle - colorless is de spindles with the sharp end;
- lymphocytes - rounded, with cytoplasm blue or gray-blue color, pronounced perinuclear area, the core of rounded, purple-pink color;
- monocytes - very large cells angularly rounded with protoplasm from bluish to greyish-blue, sometimes with vacuoles, nucleus of various shapes, sometimes eccentric located;
- platelets are small cells are oval in shape with rounded core of blue-violet color, around which is more than bright perinuclear area, and cytoplasm, painted in color from pale pink to grayish-blue.

An example of a method of staining blood samples for visualization of shaped elements.

In test tube add 0.1 ml of dye 1% brilliantissima prepared with physiological solution (pH 2,95-3), add 0.1 ml of blood and stirred for 30 s at a temperature of 18-20oWith, by gently rolling the tubes, while avoiding sudden movements.

The coloring is carried out at room temperature (18-20o(C) within 20-40 minutes. Make a smear on a glass slide and dried it. Fixed in a concentrated solution of dye-release on Leishman for 3 minutes. Without rinsing, place the swab for Domracheva the e zakreski washed smear the same buffer solution. Smear dried and carried out under the microscope immersion. For a buffer is prepared 2 solution:
1. 9.5 g of sodium disubstituted phosphate anhydrous, or 22.7 g of sodium phosphate disubstituted 12-water, brought to 1000 ml with distilled water;
2. 9,07 g potassium phosphate one-deputizing brought to 1000 ml with distilled water.

Into the flask to 1000 ml payable 63 ml of the first solution and 37 ml of the second and brought to 1000 ml with distilled water (pH of the resulting solution should be in the range of 6.8 to 7.2).

Data on microscopy are presented in tables 1-4.

Thus, one blood smear prepared on the proposed method, it is possible to conduct a series of studies that will allow us to reduce not only the time to conduct research, but also to reduce the consumption of dyes and reagents. At the same time increases the reliability of the results due to the exclusion of personal injury and damage to blood cells. Commit electron microscope stained smears allows long-term storage and re-examine them.

References
1. Antonov, B. I. and other Laboratory tests in veterinary Handbook, M., Agropromizdat, 1986, 199 S.

2. I. Bolotnikov, A. Hematology birds, Leningrad, Tashkent, Medicine, 1986


Claims

1. The visualization of blood cells of birds (platelets, reticulocytes, erythrocytes), comprising mixing the blood with the basic dye, electron microscope staining, drying is done on slides smear fixation for 3 min, counterstaining and under the microscope immersion, characterized in that the colouring unstabilized blood spend dye, brilliantissima at a temperature of 18-20C for 20-40 min, and mixing it with dye spend laminar flow in a confined space for 30 s, after drying, smear fix them in the solution-the latch on Leishman, Domracheva for 10 minutes dilute solution of dye-release on Leishman, washed with buffer solution, mikroskopiruyut.

2. The method according to p. 1, characterized in that the dilute solution of the dye-release on Leishman prepared by dilution with buffer solution under 1:1.

3. The method according to p. 1, characterized in that for the preparation of a solution of dye-release on Leishman and for rinsing strokes use a buffer solution with a pH of 6.8 to 7.2, consisting of 0,95% sodium phosphate is rastopyrivaya on smear simultaneously visualize the erythrocytes in the form of elongated ellipsoids with sharply OxyFile cytoplasm, rich in hemoglobin, the core of an intense blue-violet colour with dense chromatin structure; reticulocytes in the form of elongated ellipsoids with clearly allocated grained thread-substance in the form of blue veins on pink (exefilename) background and core from light blue to violet color; basophils in the form of dark purple cells rounded with poorly visible nuclei due to graininess, which is superimposed on the nuclear and cytoplasmic patterns; eosinophils in the form of cells rounded shape with an eccentric located light purple core and light blue cytoplasm with round grain from dark red to light pink color; pseudosinella in the form of cells rounded with a colorless segmented cytoplasm and nucleus, chromatin, light purple, light pink grain in the form of a spindle with the sharp end; the lymphocytes in the form of cells rounded, with cytoplasm blue or gray-blue color, pronounced perinuclear area, the core of rounded, purple-pink color; monocytes in the form of cells angularly rounded with protoplasm from bluish to greyish-blue, sometimes with vacuoles, nucleus of various shapes, sometimes eccentric whic is, which is more than bright perinuclear area, and cytoplasm, painted in color from pale pink to grayish blue.

 

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