RU2425370C2 - Blood, bone marrow smear, punctate and organ print staining compositions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood, bone marrow smear, punctate and organ print staining compositions contain the following ingredients: methanol - 1 litre, glycerine - 100 ml, azure2 - 12.5-15 g, eosin potassium - 8.3-10 g, potassium hydroxide - 0.15-0.23 g, citric acid - 0.18-0.26 g.

EFFECT: use of the composition provides high-quality staining of cytoplasmic and nuclear structures of all types of cells, granules of basophilic leukocytes and mast cells, granules of neutrophils, azurophilic granulosity of various cells, provides multiple use of a working staining solution for at least 1 week after preparation, ensures more efficient use of the staining agent.

Description

The invention relates to medicine and biology and can be used for microscopic examination of blood smears, bone marrow, punctate and fingerprints of various organs in the clinical laboratories of various medical institutions, as well as during scientific morphological studies in universities and research institutes of medical and biological profile.

The most common dye for staining hematological and cytological preparations currently in use is the Romanovsky-Giemsa dye. This dye was invented by the Russian doctor D.L. Romanovsky in 1891 and improved by the German chemist Giemsa in 1905 (Todorov J. Clinical laboratory research in pediatrics. Sofia: Medicine, 1963. - 874 p.). Acquaintance with the production specifications of Romanovsky dye showed that over the past 60 years they have not changed. We found that over the entire period of time, the same mistake was constantly repeated in the document TU 6-09-349-75 for the production of Romanovsky dye: along with the dosage of the dry dye of Romanovsky, methanol and glycerin, there is no dosage of the dry dye of azure 2. Therefore, various plants the amount of azure 2 is added as you wish. Only this mistake in TU explains the sharp differences in the coloring properties of different batches of Romanovsky dye.

Romanovsky dye has a lot of significant drawbacks. For each batch of dye, it is necessary to select a pH buffer for the preparation of a working solution of the dye. The working solution of the dye is unstable, so it must be prepared daily. Often, Romanovsky dye unsatisfactorily stains various cell structures: red blood cells, granularity of the three types of granulocytes, platelets, euchromatin and heterochromatin of the nuclei of cells. The dyeing time of the preparations with a working dye solution is long - 20-30 minutes. To prepare 100 ml of a working dye solution, a lot of concentrated Romanovsky dye is required - 10 or 5 ml.

The objectives of the invention are to improve the color quality of the cytoplasmic and nuclear structures of all types of cells, to ensure repeated use of the working dye solution for at least 1 week after its preparation, to increase the efficiency of dye use (increase the number of stained preparations by a dye unit by 30-50 times compared with analogues).

The tasks are achieved by increasing the concentration of ingredients in the composition, the additional introduction of buffer components. Romanovsky dye, produced by various companies, is prepared according to recipes created more than 100 years ago and includes azure-eosin according to Romanovsky (dry), azure 2, methanol and glycerin. The final concentration of azure-eosin according to Romanovsky in a standard dye is 0.76%. The compositions of the invention include the following ingredients: methanol, glycerin, azur 2, eosin-potassium, potassium hydroxide, citric acid. The quantitative ratios of the ingredients of the composition are as follows: methanol - 1 liter, glycerol - 100 ml, azure 2 - 12.5-15 g, eosin-potassium - 8.3-10.0 g, potassium hydroxide - 0.15-0.23 g , citric acid - 0.18-0.26 g. The final concentration of dyes in the compositions ranges from 2.08% to 2.5%, i.e. 2.5-3 times higher compared to the analogue.

The implementation of the invention.

To prepare composition No. 1, 1 liter of methanol is poured into a glass or plastic container, 100 ml of glycerol is added and mixed. Pour a sample of 12.5 g of azure 2 and mix thoroughly. Then, 8.3 potassium eosin is added to the same container and mixed. A container with dye solutions is placed in a thermostat with a temperature of 50-52 degrees for 18-24 hours. 0.15 g of potassium hydroxide and 0.18 g of citric acid are successively poured into heated dye solutions, stirring each time. After cooling the composition to room temperature, it is ready for use.

Preparation of composition No. 2 is carried out in a similar way, only differs in the concentration of ingredients. The quantitative proportions of the ingredients: methanol - 1 liter, glycerin - 100 ml, azur 2 - 15 g, eosin-potassium - 10.0 g, potassium hydroxide - 0.23 g, citric acid - 0.26 g.

For staining blood smears and cytological preparations from the proposed composition prepare a working solution of dye No. 1 of the following composition based on 100 ml of distilled water:

a) distilled water - 100 ml;

b) composition No. 1 - 2.5 ml.

The composition of the working solution of dye No. 2:

a) distilled water - 100 ml;

b) composition No. 2 - 2 ml.

Painting scheme:

a) fixation of smears by immersion in a solution of fixative poured into a container for 3-5 minutes;

b) swabs are quickly removed from the fixative and immediately immersed in the dye working solution (without washing);

c) washed for 2-4 s in a mixture consisting of 100 ml of distilled water and 3 ml of chilled water from a hot faucet, and dried. According to GOST, hot water has an alkaline reaction, and distilled water has a pH of 5.4-5.7 and is unsuitable for washing. Adding hot water makes distilled water neutral.

Working solutions of the proposed compositions can be used with commercially available fixatives: eosin-methylene blue according to May-Grunwald and eosin-methylene blue type Leishman. But the best results of the composition for staining hematological and cytological preparations are given with the previously proposed “Compositions for fixing hematological and cytological preparations”. This is due to the fact that the compositions for fixing and staining the drugs selected the optimal molar ratio of the main thiazine dyes and eosin, and also maintains a pH at a certain level.

The advantages of the proposed compositions for staining hematological and cytological preparations in comparison with existing analogues:

a) high quality staining of cytoplasmic and nuclear structures of all types of cells, granules of basophilic leukocytes and mast cells, granules of neutrophils, azurophilic granularity of various cells;

b) a buffer is not required for the preparation of a working dye solution; there is no need to select a pH buffer;

c) there is no need to daily prepare a working solution of the dye, because It retains coloring properties for up to 1-2 weeks;

g) economical use of the proposed composition, because 100 ml of distilled water requires only 2-2.5 ml compared to 10 and 5 ml in all analogues;

e) the proposed compositions provide coloring with one unit volume of a much larger number of drugs (30-50 times) compared with analogues.

Claims (1)

The composition for staining blood smears, bone marrow, punctate and fingerprints of various organs contains the following ingredients: methanol - 1 l, glycerin - 100 ml, azur 2 - 12.5-15 g, eosin potassium - 8.3-10 g, potassium hydroxide 0.15-0.23 g; citric acid 0.18-0.26 g.