JPH0368865A - Inspection of pathological tissue and dehydrating agent therefor - Google Patents

Abstract

PURPOSE:To perform dehydration approximately completely, to make it possible to perform delipidation and embedding thereafter in a short time and to shorten the inspecting time by performing the dehydration under the presence of specified chemical dehydration agent. CONSTITUTION:An extracted tissue undergoes fixation, dehydration and embedding. Thereafter, the tissue is cut thinly and colored. Then the tissue is inspected with a microscope. The purpose of the dehydration is to remove water which is not soluble by paraffin out of the tissue in order to ensure the permeation of the paraffin which is the necessary condition for cutting the pathological tissue sample. The dehydration is performed under the presence of at least one kind of chemical dehydrating agent which is selected among 2, 2 dimethoxypropane, ethyl orthoformate, methyl orthoacetate and ethyl orthoacetate.

Description

[Detailed Description of the Invention] [1''r View of the Invention] Technical Field> The present invention provides a method for examining pathological tissue that can qualitatively improve the quality of pathological tissue specimens and shorten the dehydration time, and a method used therefor. Regarding dehydration treatment agents.

<Prior art> When a specimen collected from a living body is subjected to a pathological histopathological examination, regardless of whether the specimen is a living body or a corpse, cells that are constituents of the tissues or organs of the specimen are examined. In order to examine the morphology of the tissue in its natural state as much as possible and make accurate pathological findings and diagnoses, artificial changes in tissue morphology should be avoided as much as possible during the preparation of the specimen. Must be avoided.

Therefore, such pathological tissue specimens are generally
It is manufactured through the general process shown in the figure. Each of these processing steps is completely unnecessary, and insufficient processing at any step will cause problems in pathological diagnosis. In particular, the perfect performance of the dehydration process determines the preservation of morphology and stainability of pathological tissue specimens.

The purpose of dehydration treatment is to remove water that is incompatible with paraffin from the tissue in order to ensure paraffin penetration, which is a necessary condition for thinly slicing pathological tissue specimens. In the treatment, dehydration must be carried out to a near complete degree.

Alcohols such as methanol and ethanol are generally used as processing agents for such dehydration processing.

The principle of dehydration treatment using alcohol is to replace (dilute) water in tissues with alcohol.

This method is only for dilution and does not eliminate the water. Once water and alcohol mix and reach equilibrium, the water concentration in the weave will not decrease any further no matter how long it takes.

Therefore, it is necessary to transfer the tissue to another high-purity alcohol for similar substitution. In order to reduce the water concentration in the tissue to the required range of 0.3 to 0.590, it is necessary to carry out serial dilution by repeating this operation in several stages.

Therefore, alcohol in the dehydration process is 5 to 6 tfJ.
The processing time occupies the largest weight in the series of processing fW of dehydration, intermediate treatment, and paraffin infiltration.

<Problem to be solved by the invention> Such dehydration treatment using alcohol has the greatest effect on shrinkage, hardening, cracking, shape, and poor staining of the obtained pathological tissue specimen, and has no effect on subsequent processing. Therefore, it required the longest time.

In addition, there were cases in which the obtained pathological histological specimens showed deformation due to young-1 shrinkage cracks.

[Summary of the invention]

Summary> The present inventor has conducted intensive research in view of the above-mentioned findings, and has found that by using a specific compound that reacts with water as a dehydration treatment agent, complete dehydration can be achieved without dehydration reaching an equilibrium state. Moreover, it was found that the structure had good shape preservation and dimensional stability, and the present invention was completed.

That is, the method for examining pathological tissue of the present invention includes fixing, dehydrating, degreasing, and embedding the extracted tissue, and then cutting it into thin slices, staining the tissue, and examining it under a microscope. , the dehydration treatment is carried out in the presence of at least one chemical dehydrating agent selected from 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate. This is the method to do so.

The dehydrating agent used in the examination of pathological tissues of the present invention is at least one chemical dehydrating agent selected from 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate. It is characterized by containing.

<Effect> The method for examining pathological tissue using the dehydration treatment agent of the present invention is as follows:
．． Taking 2-dimethoxypropane as an example, it reacts with water in the tissue and changes into acetone and methanol, so as soon as the reaction occurs, water ceases to exist, so water and alcohol are called citrus? 7i does not prevent dehydration from progressing any further.

Furthermore, the produced acetone and methanol have conventionally been used as dehydrating agents for this purpose, and their production does not have a negative effect on dehydration.

Therefore, the tissue dehydration method using the chemical dehydrating agent of the present invention is more effective than the conventional dehydration treatment using alcohol.
Since the time required for dehydration is reduced to approximately 1/3, only one dehydration tank is required.

Furthermore, since the dehydration treatment is almost complete, the subsequent degreasing treatment and embedding treatment can be carried out in a short time, so that the inspection time can be significantly shortened.

In addition, since it can be completely dehydrated, there is no tissue shrinkage, hardening, cracking, deformation, or poor staining, and the tissue has good shape retention and dimensional stability, making it a qualitatively superior pathology. Tissue specimens can be obtained.

This allows accurate pathological findings and diagnosis to be made.

[Detailed description of the invention]

[I] Pathological tissue inspection method The pathological tissue inspection in the present invention involves fixing, dehydrating, degreasing, and embedding the extracted tissue, and then cutting it into thin slices, staining them, and inspecting them under a microscope. The dehydration step is carried out in the presence of at least one chemical dehydrating agent selected from 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate. is important.

(1) Fixation treatment The main purpose of fixation treatment is to prevent autologous lysis of tissues and cells (mainly denaturation of proteins, which are their central components), and formalin aqueous solution is generally used as the treatment agent. A chemical fixative solution such as

Such a processing agent has the following properties: (a) the fixative solution quickly penetrates into the center of the tissue mass before autolysis progresses too much, and (b) the fixative solution quickly penetrates into the center of the tissue mass before autolysis progresses too much, and (b) the fixative solution penetrates into the center of the tissue mass before autolysis progresses too much. It is important that the two conditions of chemical reaction and prevention of protein denaturation in subsequent stages are met.

The excised tissue is placed in a basket or the like to allow the fixative solution to permeate therein, and then placed in a stack containing the fixative solution and immersed in the fixative solution. This process is generally carried out at a temperature of 15 to 25°C for several hours to several dozen hours.

If such fixation is insufficient, the shape preservation of each part of the tissue will be poor, and it will also cause hemolysis, poor staining, etc., which will cause problems in pathological diagnosis.

(2) Dehydration treatment The purpose of dehydration treatment is to remove 7g of insects that are incompatible with paraffin from the tissue in order to ensure paraffin penetration, which is a necessary condition for thinly slicing pathological tissue specimens. In such a dehydration process, dehydration must be carried out to a nearly complete degree.

If such dehydration treatment is insufficient, tissue shrinkage, hardening, cracking, deformation, poor staining, etc. will occur, which will impede diagnosis.

Conventionally, the chemicals used for this type of dehydration treatment include
Although alcohols such as methanol and ethanol are generally used, in the present invention, at least one alcohol selected from 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate is used. It is important to use a dehydration treatment agent described below that contains a chemical dehydration agent.

(3) Degreasing and embedding treatment At each stage of degreasing and embedding, the solvent and fat present in the tissue in the previous stage are replaced with a treatment branch such as xylene or chloroform, and The purpose is to process the tissue with paraffin and finally allow the paraffin to penetrate sufficiently into the tissue for embedding.

(4) Thin sectioning, staining, and microscopic examination of the above paraffin-embedded pathological tissue specimen.11:i;
It is cut into thin slices with a thickness of 2 to 5 μm, stained using a staining method depending on the purpose of the pathological examination, and enlarged with a microscope to examine whether abnormalities such as cancerous tissue are present.

(5) Processing tanks The above-mentioned fixation treatment or embedding process is generally performed by immersing the pathological tissue in different tanks.

In order to ensure that the processing liquid at each stage permeates through these processing tanks, the number of processing tanks is determined by arranging several tanks containing the same liquid (for example, five alcohol tanks are arranged). These processing tanks are arranged so as to constitute [11], and a lift device is installed in the center thereof. A number of baskets are suspended from the lift device, and the pathological tissues are placed in the baskets, and the baskets are raised and lowered and rotated little by little at regular intervals, so that the baskets are placed one after another in each processing pile. Soak.

Generally, fixation treatment requires 1 tank, dehydration treatment requires 5 to 6 tanks, degreasing treatment requires 2 to 3 tanks, and embedding treatment requires 3 to 4 tanks, but if the dehydration treatment agent of the present invention is used, dehydration treatment can be performed in 1 tank. It is enough.

Further, in the present invention, since the dehydration treatment is sufficiently performed, the subsequent treatment is also easy (because the cypress is easily damaged, the number of cypresses to be degreased and embedded can be reduced to 1 and 3 tanks).

Therefore, inspection time can be significantly shortened.

(II) Dehydration agent The dehydration agent used in the present invention includes 2.2
- One containing at least one chemical dehydrating agent selected from dimethoxypropane (hereinafter sometimes simply abbreviated as rDMPJ), methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate is used.

Of these, 2,2-dimethoxypropane is used, with a molecule m104.15 and a specific gravity d20-0.847.
It exhibits physical properties with a boiling point of 83°C, and under certain conditions, it reacts with water and changes into acetone and methanol as shown in the following formula. In other words, as well as decomposing the water under pressure,
It also transforms itself into a different substance. Therefore, since the dehydration of DMP is a chemical reaction between DMP and water, the dehydration treatment agent of the present invention is characterized by the fact that the water itself disappears as soon as the reaction occurs.

CHOCH3CH3 1 molecule of DMP 1 molecule of water 1 molecule of acetone 2 molecules of methanol The acid concentration is greatly involved in the above reaction between DMP and water. The higher the acid concentration, the faster the reaction will proceed. Therefore,
Adding the necessary amount of acid is extremely important; if there is not enough acid, the reaction will be slow or will not occur at all.

As the acid blended in the DMP, hydrochloric acid, para-toluenesulfonic acid, oxalic acid, etc. are generally used, and among these, it is preferable to use hydrochloric acid or oxalic acid.

In order to perform dehydration, as can be seen from the above reaction formula, theoretically, a mol amount of DMP is required to decompose 1 mol of water, but in actual dehydration, there is a margin. Therefore, it is necessary to use an amount of about 1.2 times the calculated amount by mole or more.

In a field where DMP was used in an amount 1.5 times the calculated amount, the liquid after dehydration was approximately 1:1: acetone, methanol, and DMP.
The result is a 1:1 melon mixture (weight ratio).

Here, the acetone and methanol produced are the same reagents that have traditionally been used for dehydrating living tissues.
Even if such products are generated, there is no problem in inspection.

The reaction between DMP and water itself will end the moment it encounters water if an appropriate amount of acid is added as a catalyst.

However, in order to dehydrate the tissue, it is important that the dehydration treatment liquid penetrates into the center of the NHa, so a suitable amount of time is required for dehydration.

The time for such penetration varies depending on the type, thickness, and temperature of the pathological tissue, but the time required for tissue dehydration with DMP is 3 hours without heating for a tissue 3 mm thick, but when heated to 30°C, the time required for tissue dehydration is 3 hours. If heated, dehydration will be almost complete in 1.5 to 2 hours.

This result is almost the same as the permeation rate of ethanol.

On the other hand, if you actually cut 1'11 from the finish of the specimen, 4
In the case of heating from 0 to 45°C, the dehydration time of DMP is 4-5 hours when the tissue thickness is 5 mm, but when the tissue thickness is 3 mm, the dehydration time is 2.511. If the thickness of the tissue is 1 m + i, it will take 45 minutes.

(m) Experimental Example Example 1 Fixation, dehydration, degreasing, and embedding were carried out under the following conditions using Sakura Seiki's automatic vacuum fixation and embedding device, using the extracted tissue shown below. I did it.

(Tissue used) Liver thickness 1 + nuX length 15 mm x width 25 mm Liver thickness 3 mm x length 15 questions x width 25 mm Liver thickness 5 mm x length 15 mm 25m+* (processing conditions) Fixation: 20% formalin fixation Dehydration: DMP 99.75% by weight Hydrochloric acid 0.25% by weight (virtual pH -2.2) Degreasing: Chloroform embedding: Paraffin Table 1 using the above treatment solution The process shown below was performed.

Examples 2 to 5 The same method as in Example 1 was conducted except that DMP in Example 1 was changed to methyl orthoformate, ethyl orthoformate, methyl orthoacetate, or ethyl orthoacetate.

The results are shown in Table 1.

The obtained pathological tissue specimens had good tissue morphology preservation and dimensional stability.

Comparative Examples 1 and 2 Using methanol and ethanol as dehydration treatment agents, the first
The same procedure as in Length Example 1 was carried out except that the treatments shown in the table were carried out.

The results are shown in Table 1.

In addition, the obtained pathological tissue specimen had cracks, and it was difficult to say that it was a sufficient specimen.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the order of various processes performed in the pathological tissue inspection method of the present invention.

Claims (1)

[Scope of Claims] 1. A pathological tissue inspection method in which the extracted tissue is fixed, dehydrated, degreased, and embedded, and then cut into thin slices, stained, and examined under a microscope, wherein the dehydration treatment A method for examining pathological tissue, characterized in that the method is carried out in the presence of at least one chemical dehydrating agent selected from 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate. . 2,2,2-dimethoxypropane, methyl orthoformate,
1. A dehydration treatment agent used for examination of pathological tissues, characterized by containing at least one chemical dehydration agent selected from ethyl orthoformate, methyl orthoacetate, and ethyl orthoacetate.