阅读说明：本技术 一种肝脏病理大组织的脱水方法 (Dehydration method of liver pathological large tissue ) 是由 金烁 李纯 董家鸿 卢倩 项灿宏 曾建平 王良 王思远 于劭勍 姜楠 于 2021-06-15 设计创作，主要内容包括：本发明涉及病理切片技术领域,具体涉及一种肝脏病理大组织的脱水方法。具体地,本发明基于肝门胆管癌大范围肝切除组织,提供了制备可用于诊断及科学研究的肝脏5厘米以上病理大切片的肝脏组织脱水方法。采用本发明提供的脱水方法对肝脏病理大组织进行脱水,制备得到的肝脏病理大切片,可以全景展示肝脏肿瘤周围微环境及肝脏肿瘤与周围组织的关系,用于全面临床诊断及科学研究。(The invention relates to the technical field of pathological sections, in particular to a dehydration method of large pathological tissues of liver. Specifically, the invention provides a liver tissue dehydration method for preparing pathological large-scale liver sections of more than 5cm, which can be used for diagnosis and scientific research, based on the large-scale hepatectomy tissue of the hepatoportal cholangiocarcinoma. The dehydration method provided by the invention is adopted to dehydrate the large pathological liver tissue, and the prepared large pathological liver section can display the microenvironment around the liver tumor and the relationship between the liver tumor and the surrounding tissue in a panoramic way, and is used for comprehensive clinical diagnosis and scientific research.)

1. A method of dehydrating pathological large tissues of the liver, the dehydration procedure comprising:

(1) 10% formalin for 1.7-2.2 h;

(2) 70% ethanol for 3-3.5 h;

(3) 80% ethanol for 2-2.5 h;

(4) 95% ethanol for 1.3-1.8 h;

(5) 95% ethanol for 0.8-1.2 h;

(6) 95% ethanol for 0.8-1.2 h;

(7) 100% ethanol for 0.8-1.2 h;

(8) 100% ethanol for 0.8-1.2 h;

the dehydration procedure is carried out at 32-38 ℃.

2. The dewatering method according to claim 1, wherein the dewatering procedure is:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.3-1.8 h;

(5) 95% ethanol for 0.8-1.2 h;

(6) 95% ethanol for 0.8-1.2 h;

(7) 100% ethanol for 0.8-1.2 h;

(8) 100% ethanol for 0.8-1.2 h;

the dehydration procedure was carried out at 35 ℃.

3. The dewatering method according to claim 1, wherein the dewatering procedure is:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.5 h;

(5) 1h with 95% ethanol;

(6) 1h with 95% ethanol;

(7) 100% ethanol for 1 h;

(8) 100% ethanol for 1 h;

the dehydration procedure is carried out at 32-38 ℃.

4. The dewatering method according to claim 1, wherein the dewatering procedure is:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.5 h;

(5) 1h with 95% ethanol;

(6) 1h with 95% ethanol;

(7) 100% ethanol for 1 h;

(8) 100% ethanol for 1 h;

the dehydration procedure was carried out at 35 ℃.

5. A dehydration method according to any one of claims 1 to 4 wherein said pathological large tissue of liver is hepatomegaly resected tissue of hepatoportal cholangiocarcinoma.

6. The method of claim 5, further comprising, after the dehydration procedure is completed, transparentizing the pathological large tissue of the liver with a transparentizing agent, wherein the transparentizing process comprises: xylene, 2h, 35 ℃; xylene, 2h, 35 ℃.

7. The dehydration method according to claim 6, wherein the embedding treatment is performed at 60-65 ℃ using paraffin.

8. A method for preparing a large section of a liver pathological tissue, which comprises dehydrating the liver pathological tissue by the dehydration method according to any one of claims 1 to 7.

9. A paraffin section for liver pathology, wherein the paraffin section is dehydrated by the dehydration method according to any one of claims 1 to 7; the paraffin section is a liver pathological section of more than 5 cm.

10. Use of the dehydration method according to any one of claims 1 to 7 or the preparation method according to claim 8 or the paraffin section according to claim 9 in the study of hepatoportal cholangiocarcinoma.

Technical Field

The invention relates to the technical field of pathological sections, in particular to a dehydration method of large pathological tissues of liver.

Background

The brain histopathology in the 70's of the 20 th century used the earliest pathological large section tissue to visualize the normal histology of the brain and its structures and changes. Macroscopic and microscopic features and abnormalities are realized to correlate with clinical manifestations, as well as provide information for imaging technologies emerging at the time. The application of large-section histology is of great significance to the understanding of normal anatomy of tissues, and is particularly suitable for pathologists and radiologists who are receiving training, the clinical view before diagnosis is improved by the correlation with radiographic images, and the acquisition of large pathological sections has scientific research and learning values.

For example, in the preparation of urogenital cancer specimens, the entire histopathological macro-sectioning technique can be used to compare the different structures of three prostate regions, and by means of the macro-sectioning technique, the precise information defined and forwarded to the clinician by the pathologist is: diagnostic and prognostic information, other features of clinical significance, and other features of potential clinical significance.

The liver pathological large section technology has great effect on the research of liver malignant tumors, and can be used for observing the microenvironment around the tumors in a panoramic way, positioning the relationship between the tumors and surrounding tissues and positioning the biological boundaries of the tumors. The dehydration of liver tissue is the key of the preparation link of the whole pathological large liver section, and as the liver is soft and fragile, the systematic dehydration procedure aiming at the large specimen of more than 5cm of the liver is not reported. When preparing a large pathological liver section with research value, the dehydration of the pathological liver tissue aims at preventing the shrinkage and keeping the toughness of the tissue.

Disclosure of Invention

When the large pathological liver section is prepared, the large pathological liver section contains different tissues, and the traditional section specimen preparation method cannot meet the dehydration requirement.

In order to solve the defects in the prior art, the invention aims to provide a method for dehydrating pathological large tissues of liver.

In a first aspect, the present invention provides a method for dehydrating pathological large tissues of liver, wherein the dehydration procedure in the dehydration method is as follows:

(1) 10% formalin for 1.7-2.2 h;

(2) 70% ethanol for 3-3.5 h;

(3) 80% ethanol for 2-2.5 h;

(4) 95% ethanol for 1.3-1.8 h;

(5) 95% ethanol for 0.8-1.2 h;

(6) 95% ethanol for 0.8-1.2 h;

(7) 100% ethanol for 0.8-1.2 h;

(8) 100% ethanol for 0.8-1.2 h;

the dehydration procedure is carried out at 32-38 ℃.

In the dehydration method provided by the present invention, the dehydration procedure may be:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.3-1.8 h;

(5) 95% ethanol for 0.8-1.2 h;

(6) 95% ethanol for 0.8-1.2 h;

(7) 100% ethanol for 0.8-1.2 h;

(8) 100% ethanol for 0.8-1.2 h;

the dehydration procedure was carried out at 35 ℃.

In the dehydration method provided by the present invention, the dehydration procedure may further be:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.5 h;

(5) 1h with 95% ethanol;

(6) 1h with 95% ethanol;

(7) 100% ethanol for 1 h;

(8) 100% ethanol for 1 h;

the dehydration procedure is carried out at 32-38 ℃.

The tissue penetration can be increased by treating with 70% ethanol for 3.5h, the ethanol treatment gradient is continuously increased to 80% ethanol for 2.5h, the tissue penetration can be increased, slow dehydration is performed, and tissue collapse is prevented, the 70% ethanol treatment for 3.5h and the 80% ethanol treatment for 2.5h are performed in a matching manner, so that the large liver tissue can adapt to the dehydration treatment of high-concentration ethanol, and the tissue integrity can be maintained while the dehydration is performed.

As a specific embodiment of the present invention, in the dehydration method provided by the present invention, the dehydration procedure is:

(1) 10% formalin for 2 h;

(2) 70% ethanol for 3.5 h;

(3) 80% ethanol for 2.5 h;

(4) 95% ethanol for 1.5 h;

(5) 1h with 95% ethanol;

(6) 1h with 95% ethanol;

(7) 100% ethanol for 1 h;

(8) 100% ethanol for 1 h;

the dehydration procedure was carried out at 35 ℃.

The fixed pathological tissues contain much water, and the water in the tissues must be replaced. When the water replacement is carried out on pathological large tissues, whether the dehydrating agent can enter the deep layer of the large tissues or not is considered, and the large tissues are prevented from becoming fragile due to the severe contraction effect of the dehydrating agent on the tissues.

More difficult, the large pathological tissues contain different types of cells and tissues. The invention adopts ethanol with low concentration gradually increased to high concentration as dehydration liquid to gradually replace the water in the tissue. When tissues are dehydrated in ethanol, the dehydration time needs to be accurately controlled, and particularly when the dehydration time is too short, the dehydration is insufficient, so that the next step of transparency is influenced; if the dehydration time is too long, the tissue shrinks and hardens, making it difficult to cut an ideal section.

In the invention, the dehydration process of pathological large tissues is carried out, and a good foundation is provided for the subsequent use of xylene as a clearing agent through the combination of low-concentration ethanol and high-concentration ethanol (gradient ethanol) and proper treatment time.

In the dehydration method provided by the invention, the pathological large tissue of the liver is a large-scale hepatectomy tissue of the hepatoportal cholangiocarcinoma.

The dehydration method provided by the present invention further comprises, after the dehydration procedure is completed, transparentizing the pathological large tissue of the liver with a transparentizing agent, wherein the transparentizing treatment is: xylene, 2h, 35 ℃; xylene, 2h, 35 ℃.

If the pathological tissues are in xylene, the time is too short, the section is affected after paraffin embedding, and if the tissues are in xylene, the time for transparency is too long, the tissues become brittle, and the complete section is difficult to cut.

In the dehydration method provided by the invention, after the xylene treatment is finished, paraffin embedding treatment is carried out at 60-65 ℃, and transparent pathological tissues are embedded by using paraffin for 1h, paraffin for 1h and paraffin for 1 h. During embedding, the invention finds that the tissues can shrink, become hard, become brittle and are not sliced when the temperature of the paraffin is too high or the paraffin is too long.

In a second aspect, the present invention provides a method for preparing a large section of a liver pathological tissue, in which the liver pathological tissue is dehydrated using the above-mentioned dehydration method.

In a third aspect, the invention provides a paraffin section for liver pathology, wherein the paraffin section is used for dehydrating the liver pathology tissue by using the dehydration method; the paraffin section is a liver pathological section of more than 5 cm.

According to the understanding of the skilled person, the invention also claims the application of the dehydration method or the preparation method or the paraffin section in the research of the hepatobiliary duct cancer.

The dehydration target of the pathological large section has a sufficient dehydration effect, and simultaneously, compared with the conventional specimen, the dehydration target of the pathological large section is used for preventing the whole shrinkage and the surface unevenness of the tissue to the maximum extent, if the whole shrinkage or the surface unevenness of the tissue can cause the fragmentation or the surface unevenness of the section, thereby causing the failure of digital imaging.

The invention has the beneficial effects that:

(1) the dehydration time of the tissues in the low-concentration alcohol is prolonged, the dehydration method provided by the invention is suitable for the dehydration of large pathological tissues of the liver, the low-concentration alcohol has higher penetrating power to the tissues and strong concentration alcohol, the dehydration efficiency in the liver parenchyma is effectively increased, and the action time of the high-concentration alcohol is reduced to a certain extent;

(2) the invention increases the acting time of the tissue in 70 percent and 80 percent alcohol, leads the dehydration gradient to be more moderate, avoids the tissue shrinkage caused by the long-time dehydration of the tissue in high-concentration alcohol, keeps the integrity of the section to the utmost extent, effectively keeps the flatness and the toughness of the pathological tissue, and can carry out pathological diagnosis and scientific research on a large pathological section of more than 5 cm;

(3) the invention adopts 10% neutral formalin, has high stability, has little influence on antigen and antibody, has little influence on tissue DNA and RNA, and is beneficial to immunostaining;

(4) the invention fills the gap that no reliable dehydration method exists when pathological tissue slices with more than 5cm of liver are prepared in the past;

(5) the invention solves the problems that the tissues in the large pathological liver section are soft and fragile, and the large specimen section is not easy to dehydrate;

(6) digital scanning and slide reading show that the abnormal cells in the large liver section are clearly displayed, and meanwhile, blood vessels, nerves and fibrous connective tissues around the cancer tissue can be clearly observed, and the microenvironment around the cancer and the relation with the surrounding tissues can be accurately observed; and after the liver parenchyma cells and the cell nucleuses are amplified to the designated times, the liver parenchyma cells and the cell nucleuses are clearly displayed, so that the liver pathological large tissue section prepared by the dehydration method provided by the invention can reach the diagnosis and research standards.

Drawings

Fig. 1 is a schematic diagram of liver pathological tissue sampling used in embodiment 1 of the present invention.

FIG. 2 is a graph showing the effect of preparing a dehydrated paraffin block of pathological tissues of liver in example 1 of the present invention.

FIG. 3 is a schematic view showing slicing performed by using a cherry IVS-410 slicer in example 1 of the present invention.

FIG. 4 is a scan of a large pathological section obtained in example 1 of the present invention.

FIG. 5 is a diagram of atypical cells obtained by microscopic observation of a large pathological section in example 1 of the present invention.

FIG. 6 is a diagram showing parenchymal hepatocytes obtained by microscopic observation of a large pathological section in example 1 of the present invention.

FIG. 7 is a sectional view of a dehydrated large pathological specimen in comparative example 1 of the present invention.

FIG. 8 is a microscopic observation image of a large pathological section in comparative example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

Example 1

The embodiment provides a dehydration method of a liver pathological large section with the length of more than 5cm, which comprises the following steps:

firstly, dehydration pretreatment, namely placing postoperative liver tissues (shown in figure 1) in 10 percent formalin which is 10 times of that of a specimen to be soaked for 24 hours, and using a material taking knife to take materials in a continuous radial direction with the cross section of 8-10mm (taking materials from a plane parallel to axial abdominal enhancement CT). And then, secondarily taking the specimen, and under the guidance of an embedding frame, secondarily taking the specimen by slicing at the same angle around the human body to ensure the flatness of the large specimen, wherein the tissue thickness of each slice is controlled to be 4-5mm, or the length is controlled to be 5-12cm, or the width is controlled to be 5-10 cm. The tissue was thoroughly soaked in 10% neutral formalin 10 times the tissue for 24 hours for fixation again, followed by dehydration.

The dehydration method comprises the following steps:

(1) 2h in 10% formalin, 35 ℃;

(2) 70% ethanol for 3.5h, 35 ℃;

(3) 80% ethanol for 2.5h, 35 deg.C;

(4) 95% ethanol for 1.5h, 35 ℃;

(5) 95% ethanol for 1h, 35 ℃;

(6) 95% ethanol for 1h, 35 ℃;

(7) 100% ethanol for 1h, 35 ℃;

(8) 100% ethanol for 1h, 35 ℃;

(9) xylene for 2h, 35 ℃;

(10) xylene for 2h, 35 ℃;

wax block preparation was done using 30cm circular glass petri dishes (see figure 2),

(11) paraffin wax is used for 1 hour and is 60 ℃;

(12) paraffin wax is used for 1h, 62 ℃;

(13) paraffin wax is used for 1 hour and is 60 ℃;

(14) paraffin wax was used for 1h at 62 ℃.

Slicing with cherry blossom IVS-410 slicer is completed (as shown in FIG. 3), and during slicing process, the tissue has good dehydration degree, can be completely sliced, and has slice size of above 5cm and thickness of 4 μm. And then HE staining is carried out to obtain a liver pathological large section (see figure 4), and finally a panoramic digital scanner is applied to read the section after scanning is finished.

After the film is read by a pathologist, cancer cells are clearly shown, and abnormal cells can be clearly distinguished after the amplification is carried out by 200 times. Meanwhile, blood vessels, nerves and fibrous connective tissues around the cancer tissues can be clearly observed, and the microenvironment around the cancer and the relation with the surrounding tissues can be accurately observed; the parenchymal hepatocytes and nuclei were clearly shown (see fig. 5, 6). The dehydration method provided by the invention can be universally applied to large pathological tissues of the liver.

Comparative example 1

In order to obtain a large liver tissue successfully dehydrated and used for preparing a liver pathological section of more than 5cm, the invention carries out a plurality of attempts in different improvement directions on a dehydration method, and the comparative example provides the dehydration method for preparing the large liver pathological section of more than 5cm, and is different from the example 1 in that the dehydration method adopted by the comparative example is as follows:

(1) 70% ethanol for 2 h;

(2) 80% ethanol for 1 h;

(3) 1h with 95% ethanol;

(3) 1h with 95% ethanol;

(4) absolute ethyl alcohol for 1 h;

(5) absolute ethyl alcohol for 1 h;

(6) absolute ethyl alcohol for 1 h;

(7) xylene for 05 h;

(8) xylene for 0.5 h;

(9) paraffin wax for 1 h;

(10) paraffin wax for 1 h;

(11) paraffin wax for 1 h.

In this comparative example, due to insufficient dehydration time and insufficient gradient, the dehydration time of this comparative example in low-concentration ethanol is short, and the large tissue specimen is not suitable for the dehydration of high-concentration ethanol, resulting in rapid shrinkage of tissue and surface unevenness, as shown in fig. 7.

The pathological liver section obtained by the comparative example has the defects of difficult section imaging due to the fact that the number of parenchymal cells of the liver tissue is large, the conventional dehydration gradient and the action time are insufficient, and the liver cells shrink rapidly, so that the peripheral blood vessels, nerves and fibrous connective tissues of the cancer tissue cannot be clearly observed under a microscope, and the figure 8 shows.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.