RU2550879C1 - Method of staining blood smears for microscopic determination of structural organisation of cell phase activity - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: method includes the preparation of smear from peripheral blood with preliminary fixation with methyl alcohol, drying, washing with distilled water. After that, the smears are placed in a potassium chloride solution in a ratio of 0.57 g of potassium chloride per 100 ml of distilled water for 20 min and washed with distilled water. Additionally prepared is a mixture of solutions, prepared ex tempore, containing a solution "A" and "B". The solution "A" includes a 50% silver nitrate solution in an amount of 5 g of silver nitrate + 5 ml of distilled water. The solution "B" includes a 2% solution of gelatin on a 1% formic acid solution in an amount of 15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin. The solutions "A" and "B" are mixed in an amount of 5 ml of each, in darkness, with further submergence of the blood smears for 20 min in darkness in the thermostat at a temperature of 37°C with the further submergence of the smears into distilled water for 2-3 seconds. After that, they are twice subjected to a 8 min exposure in a 5% sodium thiosulphate solution in darkness in the thermostat at a temperature of 37°C. After that, they are washed successively with tap water and distilled water, after-staining is performed in the Romanovskiy dye for 30 min. After that, the smears are washed again with tap water, air-dried, placed in the Canadian balm and covered with a coverslip.EFFECT: increased quality of smear staining and provision of a possibility to identify and further evaluate parameters of nucleolus organiser regions.4 tbl, 6 ex

Description

FIELD OF THE INVENTION

The invention relates to veterinary medicine and medicine, in particular to a method for staining blood smears for microscopic determination of the structural organization and phases of cell activity, and can be used in cytophysiology, cytopathology and cytogenetics, and is also recommended for use in hematological studies in determining the functional state of blood cells .

State of the art

There is a method of staining blood smears, which consists in the fact that for the preparation of blood smears they are fixed with a Nikiforov solution, staining is carried out with two dyes, which are taken from Ehrlich hematoxylin and eosin sodium, first the smears are placed in a solution kept in a sealed container for 360 days Ehrlich hematoxylin for 2-3 minutes, after which they are immersed in a container of tap water for 1-2 minutes until the smears turn blue, then the smears are dried with filter paper and placed in a 1% aqueous solution of eosin-sodium for 1-2 minutes, p wash the stained smears with distilled water for 8-10 s and dry in air (Patent RU No. 2304776 "Method for staining blood smears" / V.I. Trukhachev, V.V. Rodin, V.V. Mikhailenko, A.A. Dergunov / IPC G01N 33/48, G01N 1/30 publ. 08/20/2007).

The disadvantages of this method are:

- the inability to identify areas of the nucleolar organizers due to the non-specificity of the dye;

- higher complexity and economic cost of the method.

A known method of staining blood smears, which consists in the fact that the smears are fixed and stained by lowering them 4-5 times for one second in solutions of anionic dye - 0.5% eosin solution, and then in cationic dyes - 0.5% solution of azure and separately in a 0.5% solution of methylene blue. The method allows to accelerate the color of smears and improve the quality of smears for microscopic examination (Patent RU No. 2081404, “Method for staining blood smears” / S.A. Pozov, II Kolomysov / IPC, G01N 1/30, G01N 1/28, publ. 10.06.1997).

The disadvantage of this method is:

- the inability to identify areas of the nucleolar organizers, which does not allow us to judge the functional activity of cells.

A known method for staining blood smears is that blood smears are thermally fixed at 160-180 ° C, stained with 0.25% aqueous eosin solution for 110-130 s, rinsed with water, and then stained with 0.2% with an aqueous solution of diazagemicianin benzthiazole 50-70 s (Patent RU No. 2044295 “Method for staining blood smears” / V.V. Rogovin, S.I. Tsikalov, I.G. Khan, S.P. Titova // IPC G01N 1 / 30 publ. 09/20/1995).

The disadvantage of this method is the inability to identify areas of the nucleolar organizers in connection with the non-specificity of the dye.

The closest in technical essence and the achieved positive effect and adopted by the authors for the prototype is a method of staining the preparations according to the Howell and Black methods with small modifications, aimed at identifying the areas of the nucleolar organizers. Cells were fixed with a cold mixture of methanol and glacial acetic acid (3: 1) for 30 minutes. Next, the cells are washed sequentially (3 shifts): 50%, 30% ethanol, running, distilled water, dried in air. Two solutions are prepared for staining the preparations: the first includes 2% gelatin and 1% (w / w) formic acid in double-distilled water; the second - 50% AgNO ₃ in bidistilled water. On a clean glass slide apply one drop of a solution of gelatin and two drops of a solution of silver nitrate. Place the coverslip with the cells down on a drop. Staining is carried out in a humid chamber, in a thermostat at 60 ° C. Coloring time varies from 4 to 11 minutes. Staining intensity is monitored visually using an MBI-3 optical microscope (lens × 10, eyepiece × 10). Stained glass is washed with distilled water until the water becomes clear. Then, in order to avoid discoloration of the preparations over time, the glass with the cells was placed for 30 s in a 5% sodium hyposulfite solution, then the glasses were washed with water, dehydrated in alcohols and enclosed in Canadian balm (see the Howell staining method and Black, Howell M., Black DA 1980. Controlled silverstaining of nucleolus organizer regions with a protective colloidal developer. A 1-step method. Experientia. V.36, P.1014-1015).

This method of painting has the following disadvantages:

- higher complexity of operations during staining;

- has a longer coloring process;

- there is no possibility of morphological identification of cells.

Disclosure of invention

The objective of the invention is to develop a method for staining blood smears for microscopic determination of the structural organization and phases of cell activity, which has a reduction in the time to obtain large and small batches of qualitatively stained blood smears, in which it is possible to study areas of the nucleolar organizers to determine the functional activity of blood cells (see table. 1-4).

The technical result that can be achieved using the present invention is to reduce the time and improve the quality of staining of smears, with the possibility of identifying and subsequent evaluation of the parameters of the zones of the nucleolar organizers.

The technical result is achieved using the method of staining blood smears for microscopic determination of the structural organization and phases of cell activity, including the preparation of a smear from peripheral blood with preliminary fixation with methyl alcohol, drying, washing with distilled water, followed by placement in a solution of KCl in the ratio of 0.57 g KCl per 100 ml of distilled H ₂ O for 20 min, and rinsing with distilled water, while additionally preparing a mixture of solutions prepared ex tempore containing a solution "A", including 50% silver nitrate solution, in an amount of 5 g of AgNO ₃ + 5 ml of distilled water, and solution "B", including 2% gelatin solution in 1% formic acid solution in an amount of: 15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin, then mix solutions “A” and “B” in an amount of 5 ml each, in the dark, and blood smears are immersed in the resulting mixture for 20 minutes, in the dark , in a thermostat, at a temperature of 37 ° C, followed by immersion of the swabs for 2-3 s in distilled water, then incubated twice for 8 min in a 5% solution e Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C, washed sequentially with tap water and distilled water, do dyeing in Romanovsky paint for 30 minutes, washed again with tap water, dried smears in air, enclosed in Canadian balsam and covered cover glass.

Thus, the proposed method allows for hematological studies in medicine and veterinary medicine to conduct an express analysis of changes in the structure and phases of the functional activity of blood cells, give conclusions about the intensity and stability of physiological processes, to identify signs that determine the prognosis of the disease.

The essence of the method of staining blood smears for microscopic determination of the structural organization and phases of cell activity is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Place in a mixture of solutions prepared by ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 20 min, in the dark, in an thermostat, at a temperature of 37 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 8 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Carry out dye-painting with Romanovsky paint for 30 minutes. Washed with tap water and dried strokes in air. Then smears are enclosed in Canadian balsam and covered with coverslip. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

Brief description of drawings and other materials

Table 1 gives a method for staining blood smears for microscopic determination of the structural organization and phases of cell activity, the number of NLF in the nuclei of red blood cells in geese of different ages, pcs.

In table 2 - the same, the area of NLA in the nuclei of red blood cells of geese of different ages, microns square.

In table 3 - the same, the number of NLF in the nuclei of red blood cells in ducks of different ages, pcs.

In table 4 - the same, the area of NLA in the nuclei of red blood cells of ducks of different ages, microns square.

The implementation of the invention

Examples of specific implementation of the method of staining blood smears for microscopic determination of the structural organization and phases of cell activity

Example 1. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Put in a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 15 min, in the dark, in an thermostat, at a temperature of 30 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 5 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Dry smears in the air. Then smears are enclosed in Canadian balsam and covered with coverslip. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

When analyzing the data obtained, the following was noted:

- the zones of the nucleolar organizers are poorly visualized, and also were fuzzy;

- determination of the parameters of the zones of the nucleolar organizers was difficult;

- over time, the quality of the smear deteriorated, which worsened the visualization of the zones.

Example 2. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Put in a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 20 min, in the dark, in an thermostat, at a temperature of 37 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 8 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Dry smears in the air. Then enclose in a Canadian balm and cover with a cover glass. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers, while tables 1-4 provide examples of the determination of NNOR in the nuclei of red blood cells in geese of different ages, pcs .; area NNOR in the nuclei of red blood cells of geese of different ages, microns square .; the number of NLA in the nuclei of red blood cells in ducks of different ages, pcs .; area NNOR in the nuclei of red blood cells of ducks of different ages, microns square.

When analyzing the data obtained, the following was noted:

- The zones of the nucleolar organizers are visualized clearly;

- determination of the parameters of the zones of the nucleolar organizers is available in a light microscope;

- the storage time of smears did not affect the quality of visualization of the zones.

Example 3. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Stir in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Stir into a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 25 min, in the dark, in an thermostat, at a temperature of 60 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 10 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Dry smears in the air. Then enclose in a Canadian balm and cover with a cover glass. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

When analyzing the data obtained, the following was noted:

- repainting of the zones of the nucleolar organizers;

- there is excessive darkening of the image, which complicates the study of the zones of the nucleolar organizers.

Example 4. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Stir into a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 20 min, in the dark, in an thermostat, at a temperature of 37 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 8 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Post-dyeing with safranin T is carried out for 24 hours. It is washed with tap water and the smears are dried in air. Spend dehydration with alcohols. Then smears are enclosed in Canadian balsam and covered with coverslip. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

The analysis of the data showed that in the entire group of smears, 80% of the paint was washed out with alcohols, as a result of which it was not possible to identify nucleolar organizers and conduct morphological identification of blood cells.

Example 5. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Stir into a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 20 min, in the dark, in an thermostat, at a temperature of 37 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 8 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed in tap water, then with distilled water. Carry out a dye brilliant green for 2 minutes. Washed with tap water and dried strokes in air. Then smears are enclosed in Canadian balsam and covered with coverslip. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

When analyzing the data obtained, it was revealed that when the dye was brilliant green, all cell structures acquired an intensely green color. As a result, the study of the parameters of the nucleolar organizers was not possible.

Example 6. For the purpose of staining, a batch of 30 strokes was taken. Coloring is as follows. Prepared smears from peripheral blood are pre-fixed with methyl alcohol, dried under the hood and washed with distilled water. Place in a KCl solution (0.57 g KCl per 100 ml of distilled H ₂ O) for 20 minutes. Then washed with distilled water. Put in a mixture of solutions prepared ex tempore. Solution “A” - 50% silver nitrate solution (5 g of AgNO ₃ + 5 ml of distilled water). Solution “B” - 2% gelatin solution for laboratory work on a 1% solution of formic acid (15.8 ml of distilled water + 0.2 ml of 100% formic acid + 4.0 ml of 10% gelatin for laboratory work). Solution “A” (5 ml) and “B” (5 ml) are mixed in the dark, and blood smears are immersed in the resulting mixture for 20 min, in the dark, in an thermostat, at a temperature of 37 ° C. After that, the smears are immersed for 2-3 seconds in distilled water, then they are kept twice for 8 minutes in a 5% solution of Na thiosulfate, in the dark, in a thermostat, at a temperature of 37 ° C. Washed with tap water, then distilled water. Carry out dye-painting with Romanovsky paint for 30 minutes. Washed with tap water and dried strokes in air. Then smears are enclosed in Canadian balsam and covered with coverslip. Microscopy of smears is carried out in order to determine and evaluate the parameters of the zones of the nucleolar organizers.

An analysis of the data obtained led to the conclusion that 85% of the smears were successfully stained and both cell structures and nucleolar organizers can be studied in them. The proposed staining method surpasses the most commonly used staining method for detecting nucleolar organizers according to Howell and Black (see Howell and Black, 1980) in that, along with the study of nucleolar organizers, it is possible to study other cell structures, while the method significantly reduces the time spent, related to coloring.

Thus, based on the above examples, it was found that examples 2 and 6 are the most optimal, according to which it is possible to study the areas of the nucleolar organizers to determine the functional activity of blood cells, while tables 1-4 present examples of the determination of NEC in the nuclei of red blood cells in geese of different ages , PC.; area NNOR in the nuclei of red blood cells of geese of different ages, microns square .; the number of NLA in the nuclei of red blood cells in ducks of different ages, pcs .; area NNOR in the nuclei of red blood cells of ducks of different ages, microns square.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- reduction of staining time;

- the method allows to obtain strokes of high quality staining;

- the method allows to visualize the nucleolar organizers simultaneously with other cell structures.

Table 1 The number of nuclear reactors in the nuclei of red blood cells in geese of different ages, pcs. Experiment stage Experimental groups males, M ± m (n = 300) females, M ± m (n = 300) 1 day 6.22 ± 0.12 8.23 ± 0.14 ## 1 month 10.02 ± 0.12 ** 9.55 ± 0.11 ** ## 3 months 7.90 ± 0.09 ** 7.68 ± 0.08 ** 6 months 11.45 ± 0.11 ** 11.52 ± 0.10 ** 9 months 9.92 ± 0.11 ** 8.92 ± 0.09 ** ## 12 months 9.00 ± 0.08 ** 9.10 ± 0.09 Note: statistical significance of differences with an earlier period: * - p <0.05, ** - p <0.01; with males of the same term: # - p <0.05, ## - p <0.01.

table 2 Area NNOR in the nuclei of red blood cells of geese of different ages, microns square. Experiment stage Experimental groups males, M ± m (n = 300) females, M ± m (n = 300) 1 day 0.293 ± 0.009 0.404 ± 0.007 ## 1 month 0.382 ± 0.009 ** 0.425 ± 0.011 ## 3 months 0.368 ± 0.007 0.356 ± 0.010 ** 6 months 0.297 ± 0.007 ** 0.262 ± 0.008 ** ## 9 months 0.343 ± 0.012 ** 0.337 ± 0.011 ** 12 months 0.234 ± 0.008 ** 0.244 ± 0.009 ** Note: statistical significance of differences with an earlier period: * - p <0.05, ** - p <0.01; with males of the same term: # - p <0.05, ## - p <0.01.

Table 3 The number of nuclear reactors in the nuclei of red blood cells in ducks of different ages, pcs. Experiment stage Experimental groups females, M ± m (n = 300) males, M ± m (n = 300) 1 day 5.12 ± 0.14 6.14 ± 0.07 ## 1 month 7.86 ± 0.08 ** 8.75 ± 0.09 ** ## 3 months 6.37 ± 0.12 ** 6.91 ± 0.11 ** ## 6 months 10.80 ± 0.07 ** 11.74 ± 0.09 ** ## 9 months 11.14 ± 0.09 ** 10.43 ± 0.11 ** ## 12 months 11.06 ± 0.10 11.59 ± 0.13 ** ## Note: statistical significance of differences with an earlier period: * - p <0.05, ** - p <0.01; with females of the same term: # - p <0.05, ## - p <0.01.

Table 4 The area of the nuclear reactors in the nuclei of red blood cells of ducks of different ages, microns sq. Experiment stage Experimental groups females, M ± m (n = 300) males, M ± m (n * 300) 1 day 0.384 ± 0.008 0.337 ± 0.007 ## 1 month 0.664 ± 0.009 ** 0.372 ± 0.009 ** ## 3 months 0.469 ± 0.008 ** 0.527 ± 0.011 ** ## 6 months 0.431 ± 0.010 ** 0.391 ± 0.008 ** ## 9 months 0.435 ± 0.011 0.414 ± 0.010 12 months 0.378 ± 0.007 ** 0.330 ± 0.008 ** ## Note: statistical significance of differences with an earlier period: * - p <0.05, ** - p <0.01; with females of the same term: # - p <0.05, ## - p <0.01.

Claims (1)

A method of staining blood smears for microscopic determination of the structural organization and phases of cell activity, including the preparation of a smear from peripheral blood with preliminary fixation with methyl alcohol, drying, washing with distilled water, followed by placement in a solution of potassium chloride in the ratio of 0.57 g of potassium chloride per 100 ml of distilled water for 20 minutes, and rinsing with distilled water, characterized in that an additional mixture of solutions prepared ex tempore containing solution "A", including containing 50% silver nitrate solution, in an amount of 5 g of silver nitrate + 5 ml of distilled water, and solution "B", including a 2% gelatin solution in a 1% solution of formic acid in an amount of 15.8 ml of distilled water + 0.2 ml 100 % formic acid + 4.0 ml 10% gelatin, then mix solutions “A” and “B” in an amount of 5 ml each in the dark, and blood smears are immersed in the resulting mixture for 20 min in the dark in an thermostat at a temperature of 37 ° C followed by immersion of the smears for 2-3 seconds in distilled water, then incubated twice for 8 minutes in 5% solution a solution of sodium thiosulfate in the dark in a thermostat at a temperature of 37 ° C, washed sequentially with tap water and distilled water, do-dyeing in Romanovsky paint for 30 minutes, washed again with tap water, dried smears in air, enclosed in Canadian balsam and covered with a coverslip.