Method for remedying poor dehydration and dyeing of biological tissue sample
By addressing the dehydration and poor staining problems encountered by biological tissue samples during the production process, the method of reprocessing the original section and improper wax block sections was used to solve the problem of improper treatment of the dehydrator in sample production, and the dyeing effect and production quality were improved.
Patent Information
- Application Number
- CN202510133453.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-06
- Publication Date
- 2025-05-13
AI Technical Summary
During the production process of biological tissue samples, problems such as dehydration, transparency, insufficient soaking, gray-white dyeing, etc., resulting in failure of staining, especially small biopsy specimens that are difficult to remedy.
By observing the external manifestations of the sample poor problem, judging the causes, and analyzing different adverse factors, the method of reprocessing the original section and improper wax sections was adopted, including steps such as withdrawing the coverslip, counterstaining, multiple dewaxing and gradient ethanol treatment.
It effectively solves the problem of improper treatment of tissue dehydrator, improves the staining effect, and ensures the production quality of biological tissue samples.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field, in particular to a method for remedying poor dehydration and staining of biological tissue samples. Background Art
[0002] The quality of biological tissue sample preparation greatly affects whether pathologists can make correct pathological diagnoses. High-quality slide specimens should have clear nuclei and cytoplasm, distinct red and blue, and clear contrast when observed under a microscope. However, it is not easy to produce a large number of high-quality sections. In the process of routine pathology preparation, problems such as tissue dehydration, transparency, insufficient wax immersion, grayish-white staining of tissue sections, and unclear tissue structure caused by various reasons are often encountered, leading to staining failures, especially for small biopsy tissue specimens such as gastroscopy biopsy specimens and punctures. Due to the small size of the tissue, once the above problems occur, most of them cannot be re-sampled. In order to avoid such incidents from happening again, it is necessary to find out the cause of the failure in a timely manner, and actively remedy and adjust.
[0003] Based on the above problems, the present invention provides a method for remedying poor dehydration and staining of biological tissue samples.
[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the above technical defects, provide a remedial method for poor dehydration and staining of biological tissue samples, study the causes of poor dehydration and staining of different biological tissue samples, analyze different adverse factors in a targeted manner, and use different measures to reprocess the original slices and improperly processed wax block slices. It is concluded that the method of reprocessing the grayish-white original slices after removing the cover glass and re-slicing the wax blocks with improper tissue processing and then re-staining the slices can effectively solve the problem of improper processing by the tissue dehydrator.
[0006] In order to solve the above problems, the technical solution of the present invention is a method for remedying poor dehydration and staining of biological tissue samples, comprising the following steps:
[0007] Step 1: observe the external manifestations of the bad biological tissue sample problem and determine the cause of the bad biological tissue sample problem. The bad dehydration and staining problem of the biological tissue sample is mainly due to improper tissue processing by the dehydrator; improper tissue processing by the dehydrator specifically includes: white spots appearing in the dewaxing of the slices, pale and dull staining of the cell nucleus, over-staining of the cell nucleus, light staining of the eosin stain, and insufficient color separation of the cytoplasm over-staining;
[0008] Step 2: Remove the cover glass from the original slices stained gray and reprocess them;
[0009] Step 3: After re-slicing the wax block with improper tissue processing, the slices are placed in high-temperature pure water and boiled; the boiled slices are restained;
[0010] Step 4: Dewax the improperly handled wax block tissue with xylene multiple times, put it into gradient ethanol until it is hydrated, and then re-dehydrate the softened tissue.
[0011] Furthermore, the reason for the white spots appearing when the slices are dewaxed is that the baking temperature is low, the glass slides are not fully dried before dewaxing, and the slices are not kept in xylene for enough time; the solution to the white spots appearing when the slices are dewaxed is to use anhydrous ethanol to remove moisture from the glass slides, and then use xylene to deparaffinize again; return the slices to xylene for a longer stay, or replace xylene, decolorize, bleach, and re-stain.
[0012] Furthermore, the pale and dull cell nuclear staining is caused by too short a retention time of the slice in the hematoxylin staining solution, excessive oxidation of the hematoxylin staining solution, loss of staining ability, and excessive decalcification; the solution to the pale and dull cell nuclear staining is to re-stain the slice and increase the time in the hematoxylin staining solution.
[0013] Furthermore, the reasons for the over-staining of the cell nuclei are that the slices stay in the hematoxylin staining solution for too long, the slices are too thick, and the differentiation step time is too short. The solution to the over-staining of the cell nuclei is to re-slice to a suitable thickness.
[0014] Furthermore, the reason why the eosin stain is light in color is that the pH value of the eosin stain is greater than 5, too much blueing solution remains, or the sections are dehydrated in ethanol for too long after eosin staining; the solution to the light coloring of the eosin stain is that after each blueing step is completed, the weak alkaline solution used is fully washed away to ensure that there is no residual weak alkaline solution on the glass slide.
[0015] Furthermore, the reasons for the insufficient color separation of the cytoplasm over-staining are that the concentration of the eosin staining solution is too high, the slices are stained with eosin for too long, and the slices go through the ethanol dehydration step too quickly after eosin staining; the solution to the insufficient color separation of the cytoplasm over-staining is: dilute the eosin staining solution, reduce the eosin staining time, and make the slices stay in the ethanol dehydration step relatively uniform.
[0016] The advantages of the present invention compared with the prior art are:
[0017] 1. The present invention studies the reasons for poor dehydration and staining of different biological tissue samples, analyzes different adverse factors in a targeted manner, and uses different measures to reprocess the original slices and improperly processed wax block slices. It is concluded that the method of reprocessing the grayish-white original slices after removing the cover glass and re-slicing the wax blocks with improper tissue processing and then re-staining the slices can effectively solve the problem of improper tissue dehydration machine processing. DETAILED DESCRIPTION
[0018] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be described clearly and completely below.
[0019] A method for remediating poor dehydration and staining of biological tissue samples, comprising the following steps:
[0020] Step 1: observe the external manifestations of the bad biological tissue sample problem and determine the cause of the bad biological tissue sample problem. The bad dehydration and staining problem of the biological tissue sample is mainly due to improper tissue processing by the dehydrator; improper tissue processing by the dehydrator specifically includes: white spots appearing in the dewaxing of the slices, pale and dull staining of the cell nucleus, over-staining of the cell nucleus, light staining of the eosin stain, and insufficient color separation of the cytoplasm over-staining;
[0021] Step 2: Remove the cover glass from the original slices stained gray and reprocess them;
[0022] Step 3: After re-slicing the wax block with improper tissue processing, the slices are placed in high-temperature pure water and boiled; the boiled slices are restained;
[0023] Step 4: Dewax the improperly handled wax block tissue with xylene multiple times, put it into gradient ethanol until it is hydrated, and then re-dehydrate the softened tissue.
[0024] The reason for the appearance of white spots during dewaxing of sections is that the baking temperature is low, the slides are not fully dried before dewaxing, and the sections are not kept in xylene for enough time; the solution to the white spots during dewaxing of sections is to use anhydrous ethanol to remove moisture from the slides, and then use xylene to deparaffinize again; return the sections to xylene for a longer stay, or replace xylene, decolorize, bleach, and re-stain.
[0025] The reason for pale and dull nuclear staining is that the slice stays in the hematoxylin staining solution for too short a time, the hematoxylin staining solution is over-oxidized, loses its staining ability, and is over-decalcified; the solution to the pale and dull nuclear staining is to re-stain the slice and increase the time in the hematoxylin staining solution.
[0026] The reasons for over-staining of the cell nucleus are that the slices stay in the hematoxylin staining solution for too long, the slices are too thick, and the differentiation step time is too short. The solution to the over-staining of the cell nucleus is to re-slice to an appropriate thickness.
[0027] The reason why the eosin stain is light in color is that the pH value of the eosin stain is greater than 5, too much blueing solution remains, or the sections are dehydrated in ethanol for too long after eosin staining; the solution to the light coloring of the eosin stain is that after each blueing step is completed, the weak alkaline solution used is fully washed away to ensure that there is no residual weak alkaline solution on the glass slide.
[0028] The reasons for insufficient color separation of cytoplasm over-staining are that the concentration of eosin staining solution is too high, the slices are stained with eosin for too long, and the slices go through the ethanol dehydration step too quickly after eosin staining; the solution to insufficient color separation of cytoplasm over-staining is: dilute the eosin staining solution, reduce the eosin staining time, and make the slices stay in the ethanol dehydration step for a relatively uniform time.
[0029] Embodiment 1
[0030] Investigation and collection of causes of biological tissue sample staining failure: 40 pathological specimens of biological tissue sample staining failure were collected, including 10 gastroscopic biopsy specimens, 10 colonoscopy biopsy specimens, 10 endometrial specimens, and 10 lung puncture specimens. At the same time, 40 specimens with normal staining (10 wax blocks of gastroscopic biopsy specimens, 10 wax blocks of colonoscopy biopsy specimens, 10 wax blocks of endometrial specimens, and 10 wax blocks of lung puncture specimens) and 10 biological tissue sample sections stained with immunohistochemistry were collected for comparative observation.
[0031] The experimental design was divided into 7 groups: the biological tissue samples that failed to stain were the staining failure group; the wax block tissues that failed to stain were processed using 4 methods, namely treatment group 1: re-staining after slicing through the adjusted staining machine program (extending the baking time); treatment group 2: re-staining after slicing through the adjusted staining machine program (replacing new hematoxylin and eosin); treatment group 3: re-staining after slicing through the adjusted staining machine program (extending the dewaxing and dehydration time of the slices in the stainer); treatment group 4: staining after slicing through the normal staining machine program, and then adjusting the glass slides with the neutral gum concentration used for sealing; at the same time, two control groups were set up: normal biological tissue sample staining control group: paraffin specimen sections processed by normal procedures were stained by normal staining machine procedures; immunohistochemistry control group: paraffin specimens that failed to stain were sliced into biological tissue samples after the immunohistochemistry staining procedure.
[0032] Remedial methods for specimens that failed to be stained: Using the 40 specimens that failed to be stained, the 40 specimens were processed using three methods. Remedial group 1: The biological tissue sample sections that failed to be stained were soaked in xylene for 30 minutes, the cover glass covering the sliced tissue was removed, and then the sections were moved to 100%, 95%, 85%, and 70% ethanol for 3 minutes each, and the sections were washed with water for 3 minutes. The washed sections were placed in a 500ml beaker containing 300ml distilled water for water bath heating, the beaker was placed in a stainless steel pot filled with water, and heated with an electric stove or induction cooker. After the water boiled, it continued to boil for 30 minutes, the beaker was cooled to room temperature, and the sections were taken out and re-stained. Remedial group 2: The wax block was re-sliced, the tissue slices were pasted on the anti-slip slide, the slices were baked at 65℃ for 30 minutes, the conventional dewaxing was performed until hydrated, the sections were placed in a stainless steel pot filled with tap water for water bath heating and boiled for 30 minutes, and then the conventional biological tissue sample staining was performed. Remedial group 3: The wax block tissue was dewaxed with xylene multiple times, immersed in gradient ethanol until hydrated, and then the softened tissue was reprocessed, that is, the wax block tissue was processed in reverse procedure to the original tissue hydration state, and then the reagent was renewed and processed again. At the same time, the original specimen sections that failed to stain were used as the control group.
[0033] The present invention and its embodiments are described above, which is not restrictive. The drawings are only one of the embodiments of the present invention. In short, if ordinary technicians in the field are inspired by it and design structural methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.
Claims
1. A method for remediating poor dehydration and staining of biological tissue samples, characterized in that: The following steps are involved: Step 1: observe the external manifestations of the bad biological tissue sample problem and determine the cause of the bad biological tissue sample problem. The bad dehydration and staining problem of the biological tissue sample is mainly due to improper tissue processing by the dehydrator; improper tissue processing by the dehydrator specifically includes: white spots appearing in the dewaxing of the slices, pale and dull staining of the cell nucleus, over-staining of the cell nucleus, light staining of the eosin stain, and insufficient color separation of the cytoplasm over-staining; Step 2: Remove the cover glass from the original slices stained gray and reprocess them; Step 3: After re-slicing the wax block with improper tissue processing, place the slices in high-temperature pure water and boil them; Counterstaining was performed on boiled sections; Step 4: Dewax the improperly handled wax block tissue with xylene multiple times, put it into gradient ethanol until it is hydrated, and then re-dehydrate the softened tissue.
2. The method for remedying poor dehydration and staining of biological tissue samples according to claim 1, characterized in that: The reason why white spots appear when the slices are dewaxed is that the baking temperature is low, the glass slides are not fully dried before dewaxing, and the slices are not kept in xylene for enough time; the solution to the white spots when the slices are dewaxed is to use anhydrous ethanol to remove moisture from the glass slides and then use xylene to deparaffinize again; return the slices to xylene for a longer stay, or replace xylene, decolorize, bleach, and re-stain.
3. The method for remedying poor dehydration and staining of biological tissue samples according to claim 1, characterized in that: The reason for the pale and dull cell nuclear staining is that the slice stays in the hematoxylin staining solution for too short a time, the hematoxylin staining solution is over-oxidized, loses its staining ability, and is over-decalcified; the solution to the pale and dull cell nuclear staining is to re-stain the slice and increase the time in the hematoxylin staining solution.
4. The method for remedying poor dehydration and staining of biological tissue samples according to claim 1, characterized in that: The reasons for the over-staining of the cell nuclei are that the slices stay in the hematoxylin staining solution for too long, the slices are too thick, and the differentiation step time is too short. The solution to the over-staining of the cell nuclei is to re-slice to a suitable thickness.
5. The method for remedying poor dehydration and staining of biological tissue samples according to claim 1, characterized in that: The reason why the eosin stain is light in color is that the pH value of the eosin stain is greater than 5, too much blueing solution remains, or the sections are dehydrated in ethanol for too long after eosin staining; the solution to the light coloring of the eosin stain is that after each blueing step is completed, the weak alkaline solution used is fully washed away to ensure that there is no residual weak alkaline solution on the glass slide.
6. The method for remedying poor dehydration and staining of biological tissue samples according to claim 1, characterized in that: The reasons for the insufficient color separation of the cytoplasm over-staining are that the concentration of the eosin staining solution is too high, the slices are stained with eosin for too long, and the slices go through the ethanol dehydration step too quickly after eosin staining; the solution to the insufficient color separation of the cytoplasm over-staining is: dilute the eosin staining solution, reduce the eosin staining time, and make the slices stay in the ethanol dehydration step for a relatively uniform time.