RU2697475C1 - Method of preparing corneal histological preparations - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to ophthalmology and histology, and is intended for preparation of histological preparations of eye cornea. A method for preparation of histological preparations of an eye cornea involves sampling of a biomaterial and its fixation in a fixing solution, washing from a fixator, dehydration, filling into paraffin, preparation of sections and their colouring. Prior to sampling biomaterial, fixing solution is pre-introduced into anterior eye chamber. At 1.5–2.0 mm from limb, at 13–15 o'clock, corneal puncture is performed with the help of insulin syringe and fixing solution is introduced into anterior chamber of eye in amount of 0.25–0.5 ml. That is followed by enucleation of the eye, followed by taking the biomaterial for further treatment.EFFECT: using the invention enables improving quality of histological preparations of the eye cornea.3 cl, 2 ex

Description

The invention relates to medicine, in particular to ophthalmology and histology, and can be used for the preparation of histological preparations of the cornea of an animal or human eye.

The histological method of research is necessary to study the structure and function of cells in the normal state, pathology, and experimental period. For histological examination, elements of tissues and organs, in particular the cornea of the eye, are taken. However, the process of histological examination is associated with certain difficulties in the preparation of this biological material: the extremely small thickness of the cornea, the violation of the integrity of the layers of the shells of the eye when removing (enucleating) the eyeball, damage to the inner lining of the cornea. All this leads to the formation of large errors in the layered structure of the membranes of the eye and, as a result, an incorrect assessment of the degree and severity of the pathological process, so their prevention is an important technological task.

A standard method for the preparation of histological preparations is known (Eliseev V.G., Afanasyev Yu.I., Yurina N.A. and others, M., "Histology", Publishing House: Medicine, 1983, p. 9-10) , which includes the following main steps: collection of biological material and its fixation, dehydration and compaction, pouring material into paraffin, preparation and staining of sections. The action of any fixative is based on complex physicochemical processes, primarily, coagulation (coagulation) of proteins. The following are used as fixatives: simple, containing one component (formalin, alcohol, acetone) and complex, containing two or more components (Carnoy liquid: absolute alcohol, chloroform, glacial acetic acid; Zenker liquid: potassium dichromate, sodium sulfate, mercuric chloride, formalin , distilled water).

The main disadvantage of this method is the low quality of histological preparations having a layered structure due to the deformation of the biological material (biomaterial) when taking and processing with chemical reagents.

A known method of preparing histological preparations, including sampling biomaterial, fixing the latter in a solution of 10-12% acidic or neutral buffered formalin, rinsing in running water for 24 hours, followed by dehydration by passing an ascending concentration through alcohols (50%, 60%, 70 %, 80%, 90%, 96% and absolute alcohol), impregnated in solutions of aromatic carbohydrates, then in a mixture of chloroform and paraffin at 37 ° C. Then the biomaterial is waxed and slices 5-7 microns thick are made on a paraffin microtome, which are mounted on glass slides. Then the sections on the glasses are dewaxed in 2 portions of xylene, passed through descending alcohols (100%, 96%, 70% ethanol), washed with distilled water, stained, enlightened in two portions of xylene and enclosed in polystyrene or biomount (Semchenko V. V., Barashkova S.A., Nozdrin V.P., Artemyev V.N. Histological technique - 3rd ed. - Omsk - Orel, 2006. - 290 p.).

The main disadvantage of this method is the low quality of histological preparations having a layered structure due to deformation of the biomaterial during collection and processing with chemical reagents for subsequent histological examination.

A known method of preparing histological preparations, in which dehydration is carried out by four-time exposure of histological samples in acetone (R. Lilly. Pathological technique and practical histochemistry. - M .: Mir, 1969, p. 69). Dehydration in acetone occurs quickly: when using fresh acetone at each stage, it is sufficient to maintain the sample in each portion of the solvent for 20 minutes. However, using fresh solvent at each stage is expensive. Therefore, fresh acetone is used only for the fourth stage, shifting the vessels by one position; four times used acetone is purified by distillation. When dehydrated according to this scheme, the samples are kept in each portion for 40 minutes. The histological preparations obtained by this technique need constant monitoring and are accompanied by the transfer of the slice from the liquid medium to the air, which leads to undesirable deformation of the biomaterial.

A known method of preparing histological preparations, according to which for the conclusion and long-term storage of stained histological preparations, a 19-21% solution of polystyrene foam in xylene is used, stabilized by the addition of dimethyl phthalate at a rate of 1: 100. After applying a drop of the solution to the prepared slice and drying it is enclosed in a transparent and storage-stable material in the form of a film (patent RU 2117273 C1, publ. 08/10/1998). One of the disadvantages of this method is the low quality of histological preparations, since the resulting film is not durable with respect to damaging agents.

Closest to the claimed method, the prototype is a method for preparing histological preparations of the enucleated eye, including taking the material and fixing it in liquid, dehydration and pouring it into paraffin, preparing and staining sections, while before fixing the enucleated eye, its sclera is stitched with threads to a depth of about 1 mm at least in two places - from above or below, and also from the sides - from the nasal or temporal side, while using a thread of material inert to the materials used in preparing the section of the cut is linen or synthetic, while the color of the threads varies in the stitching areas, in addition, after fixing the marked eye in the liquid and filling it with paraffin, the eyes are positioned before dividing into sections taking into account its initial orientation in space, for which they focus on the above thread marks. In this case, the treatment of the enucleated eye is carried out in a standard way, using, mainly, 10% formalin as a fixative, and hematoxylin and eosin as dyes (patent RU 2525436 C1, publ. 08/10/2014).

The main disadvantage of the prototype is the low quality of the resulting histological preparations having a layered structure due to deformation of the biomaterial during collection and processing with chemical reagents for subsequent histological examination.

The objective of the invention is to ensure the preservation of the histostructure of the cornea of the eye by preventing deformation of the biomaterial in the form of delamination and bending during capture and further processing.

Effect: improving the quality of histological preparations of the cornea of the eye.

The problem is achieved by the proposed method, which consists in the following:

Before the biomaterial is taken, the fixing solution is preliminarily injected into the anterior chamber of the eye, for which, in the cornea, at 13-15 hours, 1.5-2.0 mm from the limbus, the anterior chamber of the eye is punctured (paracentesis) using an insulin syringe, while the needle section is directed upward towards the limb (facing the corneal endothelium), and a fixing solution of 0.25-0.5 ml is injected into the anterior chamber of the eye. A predominantly 10-12% solution of neutral (buffered to pH 7.0) formalin is used as the fixing solution. After 10-15 minutes (the time required to fix the posterior corneal epithelium), the eye is extracted (enucleated). The biomaterial (cornea of the eye) is taken using a trephine of the cornea. Separated from the eye biomaterial (cornea) is allowed for further processing, including fixing the biomaterial, washing it with water, dehydration, pouring in paraffin, preparing sections and staining them, which are carried out as follows.

The cornea is placed in a fixing solution (10-12% formalin solution) for 24 hours. After fixation, the cornea is washed with running water for several hours to release the rest of the fixative. Then corneal dehydration is carried out by sequential treatment with ethanol solutions with increasing concentration: 70%, 80%, 96% and 100%. Next, the cornea is placed in xylene-paraffin, then in liquid paraffin for 1 hour at 56 ° C, after cooling and hardening of the paraffin, in a known manner, a block with the cornea enclosed in it is cut out and fixed in a known manner on a wooden or plastic bar.

The preparation of corneal sections is carried out in a known manner using a microtome of standard design after securing the bars on the object holder. Then, using the feeding device, the object holder together with the bar is moved for each step by a fixed distance of 3-6 microns. A microtome knife cuts off a layer — a slice — of a given thickness from a paraffin block. After that, the slices are placed on the surface of warm water for their expansion, and then on a glass slide.

Before staining the sections, the sealing medium is removed, freed from paraffin (dewaxing) using alcohols (100%, 96%, 80%, 70% and 60%) and then using the hematoxylin and eosin staining, the contrast of the studied histological preparation is achieved.

As a result, high-quality histological preparations with a preserved histostructure are obtained, which allows a quick and reliable study.

The defining hallmarks of the proposed method, compared with the prototype, are:

- prior to the collection of biological material, a fixing solution (10-12% solution of neutral buffered formalin) is preliminarily injected into the anterior chamber of the eye, after which the eye is extracted with subsequent collection of the cornea of the eye for further processing, which allows more efficient fixation of the layered structure of the cornea and to exclude its deformation during the sampling of the material;

- for the introduction of a fixing solution into the anterior chamber of the eye, in the cornea at 13-15 hours, 1.5-2.0 mm from the limbus, a puncture is made using an insulin syringe and a fixing solution is injected in a volume of 0.25-0.5 ml , which allows to improve the quality of the histological preparation of the cornea due to experimentally selected optimal volume of the inserted fixative, sufficient to exclude deformation of the cornea during sampling and processing, as well as to increase the information content of the obtained sections of histological preparations and the accuracy of the diagnosis.

Thus, the cornea of the eye, after preliminary fixation, goes through all the stages of standard histological processing up to the production of sections and staining, completely preserving the original structure, eliminating the possibility of its deformation, without causing difficulties in making a diagnosis and determining changes.

The invention is illustrated by the following examples of specific performance of the method:

Example 1

The experiment was conducted in the morphological laboratory of the Department of General Pathology of the Surgut Medical Institute on rabbits of the breed New Zealand White. In the cornea of the rabbit’s eye, at 13 hours, 1.5 mm from the limb, the anterior chamber of the eye was punctured using an insulin syringe with a volume of 1.0 ml (the needle section is directed upward towards the limb) and 0.25 ml were injected into the anterior chamber of the eye 12 % solution of neutral buffered formalin (buffered with sodium phosphates to pH 7.0 to prevent pigmentation). After 10 minutes, the eyeball was enucleated (extracted). The biomaterial (cornea of the eye) was taken using a trephine of the cornea.

The recovered cornea was placed in a 12% neutral buffered formalin solution for 24 hours. After fixation, the cornea was washed with running water for several hours and dehydrated by treatment with ethanol solutions with increasing concentration: 70%, 80%, 96% and 100%. Then the cornea was placed in xylene-paraffin, then in liquid paraffin for 1 hour at 56 ° C, after cooling and hardening of the paraffin, a block with the cornea enclosed in it was cut in a known manner and fixed on a wooden block. Slices were prepared using a rotary microtome (Leica RM 2265) after securing the bars on the object holder. Then, with the help of a feeding device, the object holder together with the bar was moved for each step at a fixed distance of 3-6 μm and slices were obtained, which were placed on the surface of warm water for their straightening, and then on a glass slide. Before staining the sections, the sealing medium was removed, the sections were removed from paraffin using alcohols with a descending concentration of 100%, 96%, 80%, 70% and 60%, then they were washed with distilled water and then the sections were stained with hematoxylin and eosin for histological examination using light microscopy.

A histological examination revealed that the preparations (sections) have a well-preserved histostructure, there are no bends and deformation of the layers of the cornea.

It was clearly seen that the anterior corneal epithelium retained its usual structure, the anterior border membrane was clearly visualized. Corneal intrinsic matter was characterized by severe edema, the most common in the posterior third. Collagen fibers of the stroma of the cornea had an elevated crimped character. The posterior border membrane is homogeneous; its boundaries were not clearly defined. From the endothelium, pronounced proliferation of process cells was detected. A histological examination was diagnosed with corneal edema.

Example 2

The experiment was conducted in the morphological laboratory of the Department of General Pathology of the Surgut Medical Institute on rabbits of the breed New Zealand White. In the cornea of the rabbit’s eye, at 15 hours, 2.0 mm from the limbus, the anterior chamber of the eye was punctured using an insulin syringe with a volume of 1.0 ml (the needle section is directed upward towards the limb), and 0.5 ml was injected into the anterior chamber 10 % solution of neutral buffered formalin. After 15 minutes, enucleation was performed (eyeball extraction). The biomaterial (cornea of the eye) was taken using a corneal trephine apparatus.

The recovered cornea was placed in a 10% neutral buffered formalin solution for 24 hours. After fixation, the cornea was washed with running water for several hours and dehydrated by treatment with ethanol solutions with increasing concentration: 70%, 80%, 96% and 100%. Then the cornea was placed in xylene-paraffin, then in liquid paraffin for 1 hour at 56 ° C, after cooling and hardening of the paraffin, a block with the cornea enclosed in it was cut in a known manner and fixed on a plastic bar. Slices were prepared using a rotary microtome (Leica RM 2265) after securing the bars on the object holder. Then, with the help of a feeding device, the object holder together with the bar was moved for each step at a fixed distance of 3-6 μm and slices were obtained, which were placed on the surface of warm water for their straightening, and then on a glass slide. Before staining the sections, the sealing medium was removed, the sections were removed from paraffin using alcohols (100%, 96%, 80%, 70% and 60%), washed with distilled water, and then the sections were stained with hematoxylin and eosin for histological examination using light microscopy.

Histological examination clearly showed a violation of the structure of the corneal stroma, visible areas of connective tissue, destructive changes in the stroma and anterior epithelium, as well as penetration of vascularized tissue between the anterior epithelium and the anterior border plate. Diagnosed with corneal leukemia, pannus.

Using the proposed method will improve the quality of histological preparations by pre-fixing the deep layers of the cornea of the eye before removing the organ (enucleation), which will preserve its structure, shape and integrity, as well as significantly increase the information content of the obtained sections of histological preparations and increase the accuracy of the diagnosis.

Claims (3)

1. The method of preparation of histological preparations of the cornea of the eye, including sampling the biomaterial and fixing it in the fixing solution, washing from the fixative, dehydration, pouring in paraffin, preparing sections and staining, characterized in that prior to sampling the biomaterial, the fixing solution is preliminarily introduced into the anterior chamber eyes, for which 1.5-2.0 mm from the limbus, at 13-15 hours, the cornea is punctured with an insulin syringe and a fixation solution is injected into the anterior chamber of the eye in an amount of 0.25-0.5 ml, last which produce enucleated eyes followed fence biomaterial further processing. 2. The method according to p. 1, characterized in that as the fixing solution use a 10-12% solution of neutral buffered formalin. 3. The method according to p. 1, characterized in that the enucleation of the eye is carried out 10-15 minutes after the introduction of the fixing solution into the anterior chamber of the eye.