Method for preparing biological tissue samples for histopathological examinations

FIELD: medicine.

SUBSTANCE: method for preparing biological tissue sample for histopathological examination wherein the biological tissue samples are sequentially processed in a fixative solution which is presented by 10% formalin buffered by sodium phosphates; the intermediate processing takes place at room temperature. Then, the biological tissue samples are washed with water. That is followed by dehydration in more than one portions of isopropanol. The samples are cleared in two portions of the intermediates containing isopropanol and mineral oil with the first portion of the intermediate containing five portions of isopropanol and one portion of mineral oil, and with the second portion containing two portions of isopropanol and one portion of mineral oil. The intermediate processing is followed by immersing the biological tissue samples into mineral oil for clearing. The biological tissue samples are impregnated with melted paraffin added with 5% of bee wax, 0.8% of dimethyl sulphoxide and 0.5% of butyl rubber.

EFFECT: reducing labour content and time for preparing the high-quality section of the histopathological examinations with the either laboratory facilities: manual and automatic lines, in any tissue processors due to the high quality biological tissue samples having been prepared.

6 cl5

 

The present invention relates to obtaining and preparing samples for research in medicine and can be used in histological studies of biological tissue samples taken from humans or animals during surgery or autopsy.

Preparation of tissues for histological studies, typically involves soaking the sample in paraffin, necessary to obtain slices with a thickness suitable for the examination in the light microscope with transmitted light. Paraffin slice is placed on a glass slide, stained and examined under a microscope. The impregnation of biological tissues paraffin requires preliminary dehydration as water-containing fabric not impregnated with paraffin. Therefore, before impregnation with paraffin samples should:

1) keep in a state as close as possible to life and to stop the processes of autolysis - fix;

2) to remove unbound water from the tissue to dehydrate;

3) to prepare the fabric to the impregnation with paraffin to enlighten.

For the analogue of the claimed invention adopted technical solutions of the same purposes that the inventive method. A method of processing histological and biological samples by successive immersions of the samples in aqueous solution fixing substances, is izvajanja 6 shifts dehydrating liquid, the last three of which contain virtually no water (R. Lilly. Histopathological technique and practical histochemistry. - M.: Mir, 1969, p.69). As a fixing substance in a known way using a 4%aqueous solution of formaldehyde, and as a dehydrating liquids ethyl or isopropyl alcohol, or mixtures thereof. Samples sequentially incubated in three portions dewatering fluids containing water: 70% ethanol for 16 hours, 85% ethanol for 8 hours, 95% ethanol for 16 hours, and then in anhydrous liquids: 100% ethanol for 2 hours. A mixture of 100% ethanol and solvent paraffin (benzene) at a ratio of 1:1 for 1 hour is an intermediate mixture, in which both ends dehydration and begins enlightenment. Further processing is carried out in two portions of benzene for 30 minutes. And then carry out (impregnation with paraffin in several changes of paraffin). As a solvent of paraffin in addition to benzene can be used toluene, xylene, petroleum ether, carbon disulphide, chloroform, and carbon tetrachloride. In addition to the very long duration of dewatering the disadvantage of this method is that all used solvents, paraffin - hazardous substances and their harmful effect is cumulative.

Known to the persons handling the surgical biopsy and autopsy materials for microscopic examination by a Russian patent for invention №2264608 (IPC G01N 1/28, G01N 1/30, declared 26.01.2004, published 20.11.2005,), according to which transaction solutions of fabrics produced on the apparatus at-4 (universal machine for histological processing and dyeing). Fixation spend 20% formalin at a temperature of 36°C for 2 hours, washed with flowing water for 4 hours, then dehydrated in five containers with alcohol concentration of 96% (in the first tank 5 hours, the second, third and fourth tanks for 3 hours, the fifth - 5 hours). Tan in the intermediate mixture - spirit-Kiselyova solution 1 hour in xylene for 1 hour, then poured paraffin at a temperature of 36°C for 1 hour and at a temperature of 56°C, 1 hour. The disadvantage of this method is also inadequate fixation by formalin in such concentration occurs excessive formation of cross-links between protein molecules of the tissues in the surface area of the sample, which prevents uniform fixation of the sample, the long duration of the dehydration, and high toxicity of xylene, creating an environmental hazard to the environment. Xylene is a neuro - and hepatoxins poison, causes heart diseases and kidney, leukemia, has toxic effects on the bone marrow and other harmful effects on human activity [Buesa RJ, Peshkov MV. Histology without xylene. Annals of diagnostic pathology, 2009. Vol.12(4), p.246-256].

The known method functions the development of biological samples for histological studies, including dehydration of the sample in isopropyl alcohol and then waxing (application for invention of the Russian Federation No. 94036621 declared 26.09.1994, published 10.07.1996 year). Tissue samples fixed in 4% formaldehyde solution in phosphate buffer for 24 hours, washed with running water for 30 minutes. For dehydration of tissue samples sequentially incubated in 4 shifts 99% isopropyl alcohol for 3 hours in each shift. For the preservation of tissue and cellular structures of the samples in isopropyl alcohol injected surfactant (surfactant) in an amount of 0.01 to 0.5 mass parts. Dehydrated samples impregnated with paraffin exposure in two changes of molten paraffin wax for 2 hours. The disadvantage of this method is difficult to remove from fabrics isopropanol for impregnation with hot paraffin. The high gradient of the solubility parameters (9 MPa) confirms this. But regidratanty must be completely removed from paraffin, which is achieved by increasing temperature at low pressure before impregnation with paraffin

The closest analogue of the same purpose as the claimed invention is a technical solution - method of sample preparation of biological tissues for histological studies involving sequential processing samples of biological tissue with a solution retainer, rinsing the water, dehydration in several portions of isopropanol, the enlightenment in two portions intermediate mixtures containing isopropanol and mineral oil, the enlightenment in mineral oil, wetting and fill samples paraffin [Buesa RJ, Peshkov MV. Histology without xylene. Annals of diagnostic pathology, 2009. Vol.12(4), p.246-256]. In the known method, the dehydration is carried out by sequential immersion of the samples in four containers with isopropanol (1 hour each), and two tanks heated to 50°With the intermediate mixtures containing isopropanol and mineral oil (1.5 hours each), and interim mixture in the first tank has the ratio of ingredients 5:1 and the second 2:1. Then the samples are incubated in prosvetitel mineral oil for at least 2 hours, followed by soaking in paraffin at 60°C in four containers of 1 hour each.

The disadvantages of the known technical solutions, both analogue and prototype, is that if their implementation is not excluded re-staining of samples of biological tissues, which increases the effort and time to obtain high-quality sections for histological studies, and/or they all use environmentally harmful xylene or spirit-xylology solutions.

The problem to which the invention is directed, is to save labor and time is Yeni obtain high-quality sections for histological studies at any level laboratories: manual posting, automatic, vistaprocessor of any type. In addition, the object of the invention is the improvement of environmental conditions in histological laboratories and in the external environment.

The technical result, which can be obtained by carrying out the invention, lies in the exclusion of subsequent re-colouring preparations of cells and use safe for personnel and environment reagents, eliminating the harmful effects of xylene and other harmful substances in consecutive posting samples of biological tissues.

The method of sample preparation of biological tissues for histological studies involving sequential processing samples of biological tissue with a solution of fixative, rinsed in water, dehydration in several portions of isopropanol, the enlightenment in two portions intermediate mixtures containing isopropanol and mineral oil, the enlightenment in mineral oil, wetting and fill samples paraffin, is that as the latch using a 10% formalin solution, buffered with sodium phosphate, processing in the intermediate mixtures is carried out at room temperature while the first portion of the intermediate mixture contains five parts of isopropanol and one part mineral oil, and the second portion contains two parts of isopropanol and one part min is a mineral oil, samples of biological tissues impregnated with molten wax, which is added 5% beeswax, 0.8% DMSO and 0.5% butyl rubber.

The task contributes to the features that characterize the invention, in some cases, its implementation or use.

In the latch for endoscopic samples add perfume label and label color, which is used eosin.

In clamp for surgical samples add perfume label and label color, which is used methylene blue.

In isopropanol and an intermediate mixture of surface-active substance, which is used Triton X 15.

Samples of biological tissues incubated in mineral oil as prosvetitel at least 2 hours.

The impregnation with paraffin is carried out at a temperature of 60°C for 4 hours, with two tanks for 2 hours or 4 tanks 1 hour.

In the prior art unknown technical solution with the declared set of essential features of the independent claims, which confirms its compliance with the conditions of patentability - novelty.

The essential distinguishing features of the independent claims of the claimed invention to a person skilled explicitly does not follow from the prior art, which confirms the conformity of the invention to condition patentability - inventive step.

The method of sample preparation of biological tissues for histological studies can be carried out with the implementation of the specified destination as follows. For sample preparation of biological tissues for histological studies the resulting material was fixed in buffered neutral 10% formalin solution (pH 7.0) for 24 hours at room temperature or 18 hours when heated to 37°C. the volume Ratio between the retainer and the fabric is not less than 20:1. For tebufelone formalin use one - and disubstituted phosphates of sodium in amounts providing a pH of the final solution at the level of 6.8-7.4. Full recipe neutral buffered formalin: 6.5 g Na2HPO4b/W and 4 g of NaH2PO4×H2About 900 ml of distilled water, 100 ml of 37% formalin). The formalin add a color label: endoscopic samples - eosin for surgical samples of methylene blue, a fragrance label. In the latch for endoscopic samples of biological tissues add the perfume label and label color, which is used eosin. In clamp for surgical samples of biological tissues add the perfume label and label color, which is used methylene blue.

After fixation in formalin samples of biological Dane is washed in running water for 15-20 minutes to remove excess release, because precipitation of phosphates formed by the action of alcohol, pollute the pipeline wall of the working chamber and the rotary valve vistaprocessor and complicate the work on the microtome upon receipt of the slices.

Then hold dehydration of samples of biological tissues by continuous immersion of the samples in the drying fluid, the first portion contains no more than 30% water, and then drying the liquid, containing no water. As the drying fluid use isopropanol, which is soft dehydratation not sealing and not tanning tissue. To facilitate impregnation of the fabrics in it add surfactant, which is used Triton X15.

For further enlightenment samples of biological tissues in the intermediate mixtures, which are a mixture of isopropanol and mineral oil in a certain ratio. The intermediate mixture I containing 5 parts of isopropanol and 1 part of mineral oil, and an intermediate mixture II contains 2 parts of isopropanol and 1 part of mineral oil. The intermediate mixture is stable, homogeneous and transparent at room temperature and used without heating at room temperature due to the presence in their composition of surfactant brand Triton X 15, they can be added to perfume and m is TKA. After processing in the intermediate mixtures of samples of biological tissues for enlightenment immersed in mineral oil. Mineral oil is a mixture of saturated hydrocarbons, liquid at room temperature. The time of exposure to mineral oil for at least 2 hours. Samples of biological tissues without any adverse effects can be stored in mineral oil at any time: for a few days or up to several months.

After that, the samples of biological tissues impregnated with molten paraffin wax with a melting point of 52°-56°C with the addition of 5% beeswax, 0.8% dysteleological facilitating the impregnation of the samples of biological tissues, and 0.5% butyl rubber, providing most of their plasticity. The impregnation with paraffin is carried out at a temperature of 60°C for 4 hours, with two tanks for 2 hours or 4 tanks 1 hour.

The duration of the posting samples of biological tissues of solutions is determined by the type of samples and their sizes. Samples having a size of 0.1×0.1×0.1 cm are scheduled for endoscopic samples, and large - scheduled for conventional surgical samples.

For surgical specimens fixation carry out the above method. Dehydration carry out sequential immersion in five tanks with isopropanol with lyderic the nd every 1 hour. For the enlightenment of their consistently immersed in two containers with intermediate mixtures in each for 1.5 hours, and then in mineral oil not less than 1.5 hours. The impregnation with paraffin is carried out at a temperature of 60°C for 4 hours (in two tanks for 2 hours or 4 tanks 1 hour). The total time of the transaction without regard to the fixation of 13.5 hours.

Fixation endoscopic samples with dimensions of 0.1×0.1×0.1 cm exercise as well as surgical specimens. Dehydration carry out sequential immersion in two shifts of a solution of isopropanol to extract each for 15 minutes. For the enlightenment of their consistently immersed in two containers with intermediate mixtures, each for 30 minutes, and then in mineral oil for 30 minutes. The impregnation with paraffin is carried out at a temperature of 60°C in two portions over 15 minutes. The total time of the transaction without regard to the fixing of 2 hours and 30 minutes.

Both schedules are suitable for use as a manual transaction, and for use in processors carousel and a vacuum type.

The method is illustrated by examples.

Example 1.

Manual posting of a sample of breast tissue, 1.5×1.5×0.3 see

1. 10% formalin, buffered with phosphates, 24 hours, room temperature, 24 hours. The ratio of the catch: the fabric is not less than 20:1.

2. Rinse in running water for 20-30 minutes.

3. 70% isopropanol for 1 hour.

4. 80% isopropanol for 1 hour.

5. 95% isopropanol for 1 hour.

6. 99.7% isopropanol for 1 hour.

7. 99.7% isopropanol for 1 hour.

8. The intermediate mixture I, 1.5 hours.

9. The intermediate mixture II, 1.5 hours.

10. Mineral oil 1.5 hours.

11. Paraffin, 60°C, 1 hour.

12. Paraffin, 60°C, 1 hour.

13. Paraffin, 60°C, 1 hour.

14. Paraffin, 60°C, 1 hour.

Fill in paraffin.

Example 2.

Manual posting sample of tissue of the stomach, the rate of 0,1×0,1×0,1, see

1. 10% formalin, buffered with phosphates, 24 hours, room temperature, 24 hours. The ratio of the catch: the fabric is not less than 20:1.

2. Rinse in running water for 5 minutes.

3. 70% isopropanol for 15 minutes.

4. 99.7% isopropanol for 15 minutes.

5. The intermediate mixture I, 30 minutes.

6. The intermediate mixture II, 30 minutes.

7. Mineral oil 30 minutes.

8. Paraffin, 60°C, 30 minutes.

9. Paraffin, 60°C, 30 minutes.

Fill in paraffin.

Describes the tools and methods by which the possible implementation of the method of sample preparation of biological tissues for histological studies, with the implementation of the specified destination, confirms the compliance of the claimed invention to the condition of patentability of industrial applicability.

1. The method of sample preparation of biological tissues for histological studies involving sequential processing samples of biological tissue with a solution of the latch, promilk is in the water, dehydration in several portions of isopropanol, the enlightenment in two portions intermediate mixtures containing isopropanol and mineral oil, the enlightenment in mineral oil, wetting and fill samples paraffin, characterized in that as the latch using a 10%formalin solution, buffered with sodium phosphate, processing in the intermediate mixtures is carried out at room temperature, while the first portion of the intermediate mixture contains five parts of isopropanol and one part mineral oil, and the second portion contains two parts isopropanol and one part mineral oil, samples of biological tissues impregnated with molten wax, which is added 5% bee wax, 0.8% of dimethyl sulfoxide and 0.5% butyl rubber.

2. The method according to claim 1, characterized in that the latch for endoscopic samples of biological tissues add the perfume label and label color, which is used eosin.

3. The method according to claim 1, characterized in that the retainer for surgical samples of biological tissues add the perfume label and label color, which is used methylene blue.

4. The method according to claim 1, characterized in that in isopropanol and an intermediate mixture of surface-active substance, which is used Triton X15.

5. The method according to claim 1, trichosis fact, samples of biological tissues incubated in mineral oil as prosvetitel at least 2 hours

6. The method according to claim 1, characterized in that the impregnation with paraffin is carried out at a temperature of 60°C for 4 h, with two tanks for 2 h or 4 tanks and 1 o'clock



 

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