阅读说明：本技术 一种肝脏病理大切片的he染色方法 (HE staining method for liver pathological large section ) 是由 金烁 李纯 曾建平 姜楠 于劭勍 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切片的HE染色方法。具体地,采用本发明提供的HE染色方法基于肝门胆管癌大范围肝切除组织,制备得到可供诊断及科学研究的5厘米以上肝脏组织病理大切片。采用本发明提供的HE染色方法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 an HE staining method for a large pathological section of liver. Specifically, a large pathological section of the liver tissue with the size of more than 5cm for diagnosis and scientific research is prepared by adopting the HE staining method provided by the invention based on the large-scale hepatectomy tissue of the hepatoportal cholangiocarcinoma. The large pathological liver section prepared by the HE staining method provided by the invention can display the microenvironment around the liver tumor and the relationship between the liver tumor and the surrounding tissues in a panoramic way, and is used for comprehensive clinical diagnosis and scientific research.)

1. An HE staining method for pathological large liver sections is characterized in that the HE staining method comprises the following steps:

xylene (I), 10min,

xylene (II), 10min,

xylene (III), 5 min;

the xylene removal treatment comprises the following steps:

100% ethanol (I), for 5min,

100% ethanol (II), 5min,

95% ethanol for 5 min;

80% ethanol for 5 min;

dyeing and differentiating by treating with 1% hydrochloric acid ethanol for 20-40 s;

eosin staining is 0.5% aqueous eosin staining for 1-3 min.

2. The HE staining method according to claim 1, wherein the time for hematoxylin staining in the HE staining method is 8 to 12 min.

3. The HE staining method of claim 2, wherein 1% ammonia is used to turn blue for 30 s.

4. The HE dyeing method according to claim 3, further comprising a post-dyeing dehydration and clearing process, the post-dyeing dehydration and clearing process being:

80% ethanol, 3min, 95% ethanol, 5min, anhydrous ethanol (I), 5min, anhydrous ethanol (II)5min, xylene (I), 5min and xylene (II), 5 min.

5. The HE staining method of any of claims 1-4, wherein the pathological large tissue of the liver is a large hepatectomy tissue of the hepatoportal cholangiocarcinoma.

6. The HE staining method of claim 5, wherein the HE staining method is:

(1) xylene (I), 10min, xylene (II), 10min, xylene (III), 5 min;

(2) 100% ethanol (I), 5min, 100% ethanol (II), 5min, 95% ethanol, 5min, 80% ethanol, 5min, washing with running water, 2min, and distilled water for 2 min;

(3) staining with hematoxylin for 10min, and washing with running water for 3 min;

(4) washing with 1% hydrochloric acid ethanol for 30s for 1 min;

(5) returning blue with 1% ammonia water for 30s, and washing with running water for 1 min;

(6) dyeing with 0.5% water-based eosin solution for 1-3min, and washing with running water for 1 min;

(7) 80% ethanol, 3min, 95% ethanol, 5min, anhydrous ethanol (I), 5min, and anhydrous ethanol (II) for 5 min;

(8) xylene (I), 5min, xylene (II), 5 min.

7. A method for preparing a large section of a liver pathological tissue, which comprises staining the liver pathological tissue by the HE staining method according to any one of claims 1 to 6, followed by sealing with a neutral gum to minimize air bubbles and obtaining a large section of the stained liver pathological tissue.

8. A paraffin section for liver pathology, wherein the paraffin section is stained for the liver pathology using the HE staining method of any one of claims 1 to 6; the paraffin section is a liver pathological section of more than 5 cm.

9. Use of the HE staining method according to any one of claims 1 to 6 or the preparation method according to claim 7 or the paraffin section according to claim 8 for the diagnosis of post-operative pathologies of liver diseases.

10. Use of the HE staining method according to any one of claims 1 to 6 or the preparation method according to claim 7 or the paraffin section according to claim 8 in the study of hepatoportal cholangiocarcinoma.

Technical Field

The invention relates to the technical field of pathological sections, in particular to an HE staining method for a large pathological section of liver.

Background

The use of histology on large sections of pathology began in the 20 th century in the 70's brain histopathology. The reason for using pathologically large sections is to visualize the normal tissue of the brain and its structure and changes, which can correlate macroscopic and microscopic features and abnormalities of the brain with clinical manifestations, including providing information for imaging techniques emerging at the time.

The application of histopathology on large sections of urogenital cancer specimens is for the same reason as that of neuropathologists. For example, the use of large-slice histology is important to understand the normal anatomy of the prostate, especially for pathologists and radiologists who are trained to improve the "clinical view" of the clinician before diagnosis by correlation with radiographic images.

The precise information that a pathologist can define and forward to a clinician by the pathology gross section technique is: diagnostic and prognostic information, including tumor multifocal and location, histological type and variation, grade, TNM staging, including surgical margin status (R) and Lymphatic Vascular Infiltration (LVI), and tumor volume and size. Other features of clinical significance and other features of potential clinical significance can also be known by the technique of pathological gross section.

The liver pathological large section technology has great effect on the research of liver malignant tumor. The technology can be used for observing the microenvironment around the tumor in a panoramic way, positioning the relation between the tumor and surrounding tissues and the biological boundary of the tumor, but at present, no research-type paper report of systematic liver pathology large sections exists internationally.

HE staining of liver tissue is the key of the whole preparation process of pathological large liver sections. The liver is the largest parenchymal organ of the abdominal cavity, in addition, the number of parenchymal cells of the liver is large, the Glisson system in the liver is complicated and complicated, the shapes of all systems and cells in the liver need to be observed simultaneously, the steps and the difficulty of HE staining are greatly different from those of other tissues, and the high requirements on the quality of HE staining are provided.

Disclosure of Invention

In order to solve the problem that the conventional HE staining is not suitable for preparing pathological large sections, the invention provides an HE staining method suitable for liver pathological large sections. The invention aims to provide an HE staining method for preparing pathological liver tissues of pathological sections of more than 5cm, which is favorable for application in postoperative pathological diagnosis and scientific research of liver diseases.

In a first aspect, the present invention provides an HE staining method for a pathological large liver section, wherein a dewaxing method in the prior art cannot be applied to dewaxing a large specimen tissue because the wax content of the large specimen tissue of more than 5cm of a liver is high, and therefore, in the HE staining method, the hydration dewaxing treatment comprises:

xylene (I), 10min,

xylene (II), 10min,

xylene (III), 5 min;

the xylene removal treatment used:

100% ethanol (I), for 5min,

100% ethanol (II), 5min,

95% ethanol for 5 min;

80% ethanol, 5 min.

Because the liver tissue specimen is larger, if hydration treatment in the conventional dyeing step is adopted, more cells in the large liver tissue have poor coloring effect due to over expansion, and observation is influenced; and the volume of the large tissues of the liver expands rapidly, which can cause the surface of the tissues to generate concave-convex and never cause the failure of digital scanning imaging. In the hydration of large tissues, the use of three-stage alcohol concentration gradients and increased alcohol exposure times are essential.

Dyeing and differentiating treatment by using 1% hydrochloric acid for 20-40 s;

eosin staining is 0.5% aqueous eosin staining for 1-3 min.

In the dyeing process, the reason for using the aqueous eosin solution is that after dewaxing and xylene removal treatment, large tissues of the liver are hydrated, the combination degree of the alcohol-soluble eosin in water is poor, the alcohol must be quickly added after dyeing is finished, and if the dyeing is slow, the color is dropped.

In the HE staining method provided by the invention, the staining time of hematoxylin is 8-12 min.

In the HE dyeing method provided by the invention, the time for returning blue by using 1% ammonia water is 30 s.

The HE dyeing method further comprises dehydration treatment after dyeing, wherein the dehydration and transparent treatment after dyeing comprises the following steps:

80% ethanol, 3min, 95% ethanol, 5min, anhydrous ethanol (I), 5min, anhydrous ethanol (II)5min, xylene (I), 5min, and xylene (II), 5 min.

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

Specifically, as a specific embodiment of the present invention, the HE dyeing method is:

(1) xylene (I), 10min, xylene (II), 10min, xylene (III), 5 min;

(2) 100% ethanol (I), 5min, 100% ethanol (II), 5min, 95% ethanol, 5min, 80% ethanol, 5min, washing with running water, 2min, and distilled water for 2 min;

(3) staining with hematoxylin for 10min, and washing with running water for 3 min;

(4) washing with 1% hydrochloric acid ethanol for 30s for 1 min;

(5) returning blue with 1% ammonia water for 30s, and washing with running water for 1 min;

(6) dyeing with 0.5% water-based eosin solution for 1-3min, and washing with running water for 1 min;

(7) 80% ethanol, 3min, 95% ethanol, 5min, anhydrous ethanol (I), 5min, and anhydrous ethanol (II) for 5 min;

(8) xylene (I), 5min, xylene (II), 5 min.

In a second aspect, the invention provides a method for preparing a large liver pathological tissue section, wherein after the liver pathological tissue is stained by the HE staining method, the large liver pathological tissue section is sealed by neutral gum, so that bubbles are reduced as much as possible, and the large stained liver pathological tissue section is obtained.

In a third aspect, the present invention provides a paraffin section for liver pathology, wherein the paraffin section is used for staining the pathological tissues of the liver by using the above HE staining 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 above HE staining method or the above preparation method or the above paraffin section in the pathological diagnosis after liver disease operation and the study of hepatoportal cholangiocarcinoma.

The invention has the beneficial effects that:

(1) the invention can fully dewax the tissue of a large sample with the size of more than 5cm by increasing the acting time of the dimethylbenzene, so that the tissue fully enters a water-soluble state;

(2) in the hydration process, the action time of 100 percent alcohol is increased, and the long-time hydration steps of 95 percent and 80 percent alcohol are also increased, so that the concentration and the action time of the alcohol used for treatment are increased, the hydration gradient of liver tissues is more moderate, and the cells are fully prevented from over-expansion;

(3) the aqueous eosin solution adopted by the invention is easy to combine with hydrated large specimen tissues, and the staining is more stable in HE staining of large specimens;

(4) the invention provides a method for staining pathological tissues of liver, wherein a large pathological section prepared by the staining method is suitable for the diagnosis-level research of liver diseases, the cell nucleuses of various cells in the pathological section are clearly displayed, and the cells and tissues are uniformly stained;

(5) the large pathological liver section prepared by the HE staining method can clearly distinguish the accessory nerves, arteries, veins and fibrous connective tissues of the liver under a microscope, and is favorable for observing the biological behavior of tumors.

Drawings

FIG. 1 is a diagram of pathological tissues of liver used in example 1 of the present invention.

FIG. 2 is a diagram of atypical cells obtained by microscopic observation of a large pathological section in example 1 of the present invention.

FIG. 3 is a diagram of parenchymal hepatocytes obtained by microscopic observation of a large pathological section in example 1 of the present invention.

FIG. 4 is a microscopic view 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

This example provides a method for HE staining of pathological tissues to prepare large pathological sections of 5cm or more.

First, HE dyeing pretreatment

As shown in figure 1, the liver tissue after operation is soaked in 10% formalin 10 times of the specimen for 24 hours, and then the material is obtained by using a material-obtaining knife in a continuous radial direction with the cross section of 8-10mm thickness (the 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 4-5 mm. Fully soaking the tissues in 10% neutral formalin which is 10 times of the tissues, fixing for 24 hours again, and entering an HE staining stage after the dehydration machine baking procedure is completed.

Secondly, the HE dyeing method comprises the following steps:

after coloring is finished, the invention adopts 80 percent, 95 percent and absolute ethyl alcohol gradient alcohol to dehydrate in 18 to 21 steps, and controls dehydration time, so that dehydration process is more moderate, and pathological observation is effectively prevented from being influenced by rapid shrinkage of cells of large slices. Meanwhile, the occurrence of unevenness on the surface of the tissue is prevented, so that the digital imaging fails.

And finally, using an Olympus VS200 panoramic digital scanner to read the film after scanning is finished.

After the section is read by a pathologist, the whole liver tissue of the large liver tissue section prepared by the embodiment is uniformly colored, no obvious color difference is seen, hepatocellular carcinoma cells are clearly displayed after further amplification, abnormal cells (shown in figure 2) can be clearly distinguished after 200 times of amplification, 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. The parenchymal hepatocytes and nuclei were clearly shown (see FIG. 3). The HE staining method can be universally applied to large pathological tissues of the liver.

After the section is read by a pathologist, the whole liver tissue of the large liver tissue section prepared by the embodiment is uniformly colored, no obvious color difference is seen, hepatocellular carcinoma cells are clearly displayed after further amplification, abnormal cells can be clearly distinguished after 200 times of amplification, blood vessels, nerves and fibrous connective tissues around the cancer tissue can be clearly observed, and the microenvironment around the cancer and the relation between the microenvironment around the cancer and the surrounding tissues can be accurately observed. Parenchymal hepatocytes and nuclei were clearly shown. The HE staining method can be universally applied to large pathological tissues of the liver.

Comparative example 1

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

(1) 3min of dimethylbenzene;

(2) 3min of dimethylbenzene;

(3) 3min of dimethylbenzene;

(4) absolute ethyl alcohol for 3 min;

(5) 95% ethanol for 3 min;

(6) 95% ethanol for 3 min;

(7) 80% ethanol for 3 min;

(8) 70% ethanol for 3 min;

(9) 1min tap water;

(10) 7min of hematoxylin;

(11) tap water lmin;

(12) 0.5-1% hydrochloric alcohol < 75% alcohol > differentiation solution for 30 s;

(13) 1min tap water;

(14) returning the 0.5-1% ammonia water to the blue solution for 30 s;

(13) tap water lmin;

(14) 0.5% alcohol < 80% ethanol > soluble eosin 30 s;

(15) 70% ethanol for 30 s;

(16) 80% ethanol lmin;

(17) 95% ethanol for 2 min;

(18) 95% ethanol for 2 min;

(19) absolute ethyl alcohol for 1 min;

(20) 3min of dimethylbenzene;

(21) 3min of dimethylbenzene;

(22) 3min of dimethylbenzene;

(23) and (5) entering a mounting machine for mounting neutral gum.

The microscopic observation picture of the large section obtained by the comparative example is shown in figure 4, and the staining program of the comparative example can obtain the large pathological section of the liver which is not uniformly stained, can not accurately distinguish the cell nucleus of various cells, and declares the staining failure.

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.