CN113790946A - Intercellular substance staining kit for digital pathological scanning analysis system - Google Patents
Intercellular substance staining kit for digital pathological scanning analysis system Download PDFInfo
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Abstract
The invention discloses a cell stroma staining kit for a digital pathology scanning analysis system, which comprises hematoxylin staining solution, eosin staining solution and differentiation solution, wherein each liter of hematoxylin staining solution contains the following components: 1.9-4.0 g of hematoxylin, 15-25 g of aluminum potassium sulfate, 3.5-4.5 g of sodium iodate, 180-220 mL of glycerol, 5-15 mL of ethanol and the balance of water; every liter of eosin dye solution contains the following components: 7.5-12.5 g of eosin Y, 900-980 mL of ethanol and the balance of water; each liter of differentiation solution contains the following components: 650-800 mL of ethanol, 0.35-0.55 g of HCl and the balance of water. The dyeing reagent of the formula has the characteristics of strong tinting strength, bright color, bright contrast, uniform color and long-term storage, and can meet higher requirements of a digital pathological scanning analysis system.
Description
Technical Field
The invention relates to a intercellular substance staining kit, in particular to a intercellular substance staining kit for a digital pathology scanning analysis system.
Background
The tumor stroma is composed of cancer-associated fibroblasts, endothelial cells, amyloid cells, pericytes, lymphocytes, and extracellular matrix. The tumor microenvironment plays an important role in the growth process of tumors. The tumor stroma such as fibroblast, endothelial cell, inflammatory cell, pericyte and extracellular matrix plays a decisive role in the biological behavior (growth, infiltration, metastasis and the like) of the tumor.
The tumor-stromal ratio (TSR), the ratio of tumor cells to the stromal fraction within tumor tissue, has been shown in several previous studies to be: TSR is an independent prediction factor for prognosis of breast cancer, cervical cancer, esophageal cancer and other malignant tumor patients.
The evaluation of the tumor-to-stroma ratio in the current study is mainly performed by staining postoperative pathological sections and then manually calculating TSR values by experienced pathologists. Among them, hematoxylin-eosin (HE) staining is a commonly used staining method due to its easy operation and low cost. The dyeing is carried out according to the principle that different components of tissues or cells have different affinities and different dyeing properties for basic dye (hematoxylin) and acid dye (eosin), cell nuclei and the like consisting of acid substances are dyed into distinct bluish purple by the hematoxylin, and various components containing the basic substances, such as cytoplasm, muscle fibers, collagen fibers and the like, show red with different depths. However, the result has very large uncertainty and non-repeatability by manually calculating the TSR value, 10% is used as a value interval at present, TSR calculation within the range of 40% -60% is a difficult point for evaluation, and the accuracy of TSR evaluation can be greatly improved by adopting digital image analysis equipment.
The conventional hematoxylin staining solution is usually prepared by a Harris formula or a Drill formula, but the staining solution prepared by the Harris formula has a good staining effect only when the preparation is finished, is easy to generate precipitates and gradually loses the staining effect after being placed for a long time, is relatively difficult to generate precipitates in the Drill formula, and has a poor effect in the analysis of a digital image with higher requirements on color vividness, contrast and accuracy.
Therefore, it is a trend to design HE staining kits dedicated to digital image analysis devices in order to make the experimental procedure more convenient, accurate and fast.
Disclosure of Invention
In order to achieve the above object, the present invention provides a intercellular substance staining kit for a digital pathology scanning analysis system, comprising a hematoxylin staining solution, an eosin staining solution and a differentiation solution; wherein the content of the first and second substances,
each liter of hematoxylin staining solution contains the following components: 1.9-4.0 g of hematoxylin, 15-25 g of aluminum potassium sulfate, 3.5-4.5 g of sodium iodate, 180-220 mL of glycerol, 5-15 mL of ethanol and the balance of water;
every liter of eosin dye solution contains the following components: 7.5-12.5 g of eosin Y, 900-980 mL of ethanol and the balance of water;
each liter of differentiation solution contains the following components: 650-800 mL of ethanol, 8-17 mmol of HCl and the balance of water.
Preferably, the content of the hematoxylin in each liter of hematoxylin staining solution is 2.2-3.6 g.
Preferably, the content of the aluminum potassium sulfate in each liter of hematoxylin staining solution is 18-22 g.
Preferably, the content of sodium iodate in each liter of hematoxylin staining solution is 3.8-4.2 g.
Preferably, the eosin Y content per liter of eosin staining solution is 8.0-12.0 g.
Preferably, the content of the ethanol in each liter of the differentiation solution is 650-750 mL.
Optionally, the hematoxylin staining solution further contains glacial acetic acid.
Optionally, the eosin dye solution also contains glacial acetic acid.
Further, the hematoxylin stain of the present invention is not required to be stored in a brown reagent bottle away from light, and in a preferred embodiment of the present invention, the hematoxylin stain, eosin stain and differentiation solution are stored in conventional white or milky reagent bottles.
Preferably, the reserves of the hematoxylin staining solution, the eosin staining solution and the differentiation solution in the kit are equal.
Because the pathological section staining quantity of the hospital is large, the kit is convenient to take when designing the packaging specification, and the quantity of samples is required to be met as far as possible, a dye vat for soaking the section in the pathology department can be generally used for 5 pathological sections at the same time, 50 mL of reagents are required, namely 10 mL of reagents are required for each section sample, each reagent in the kit can adopt the reagent quantity corresponding to 10 parts, 15 parts, 20 parts, 25 parts or 30 parts of pathological sections as a package, and correspondingly, the reagent quantity stored in each reagent bottle is preferably 110-150 mL, 160-200 mL, 210-250 mL, 260-300 mL or 310-350 mL.
Preferably, the kit provided by the invention is stored at the temperature of 10-30 ℃ for 6-24 months.
Further, the kit of the present invention further comprises a blocking agent; preferably, the blocking agent is Eukit.
Further, the kit of the invention also comprises one or more of absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol and xylene; preferably, the reserves of the above reagents are twice of the hematoxylin staining solution.
Further, the kit of the present invention further comprises a cover slip and/or a slide.
Further, the kit of the invention also comprises an instruction for use.
Further, the invention provides a preparation method of hematoxylin staining solution, which comprises the following steps:
1) dissolving hematoxylin with anhydrous ethanol;
2) dissolving aluminum potassium sulfate in water;
3) mixing the ethanol solution of the hematoxylin obtained in the step 1) with the aqueous solution of the potassium aluminum sulfate obtained in the step 2) and the sodium iodate;
4) adding glycerol into the aluminum potassium sulfate aqueous solution obtained in the step 2) or the mixed solution obtained in the step 3).
Preferably, the step 2) can be heated to promote dissolution, and the heating temperature is preferably 80-90 ℃.
In one embodiment of the present invention, the sodium iodate is added to the aqueous solution of the aluminum potassium sulfate obtained in the step 2), the ethanol solution of the hematoxylin obtained in the step 1) is added thereto, and finally the glycerol is added and then mixed uniformly in the steps 3) and 4).
In another embodiment of the present invention, the glycerol in steps 3) and 4) is added to the aluminum potassium sulfate aqueous solution obtained in step 2), and then the ethanol solution of the hematoxylin obtained in step 1) is added thereto and mixed uniformly.
Preferably, the preparation method further comprises adding glacial acetic acid into the mixed solution.
Further, the invention provides a preparation method of eosin dye solution, wherein the eosin dye solution is prepared by directly dissolving eosin Y in ethanol solution, and the ethanol solution can be any one of 98% ethanol, 95% ethanol, 92% ethanol and 90% ethanol.
In other embodiments, the eosin dye solution is dissolved by water containing glacial acetic acid, and then ethanol or ethanol solution is added, wherein the ethanol solution can be any one of 98% ethanol, 95% ethanol, 92% ethanol and 90% ethanol.
Further, the invention provides a preparation method of the differentiation solution, namely dripping concentrated hydrochloric acid into an ethanol solution according to the calculated amount, wherein the ethanol solution can be any one of 80% ethanol, 75% ethanol, 70% ethanol and 65% ethanol.
Further, the invention also provides a intercellular substance staining method when the kit is used in a digital pathology scanning analysis system, which comprises the following steps:
i) dewaxing: soaking and dewaxing by using dimethylbenzene, and then sequentially soaking and washing by using absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol and water;
ii) dyeing: sequentially soaking and dyeing with hematoxylin dye solution, soaking and differentiating with differentiation solution, washing with running water, soaking with 80% ethanol, and soaking and dyeing with eosin dye solution;
iii) dehydration: soaking in 80% ethanol, 95% ethanol, and anhydrous ethanol sequentially for dehydration;
iv) transparency: soaking in xylene to make it transparent;
v) sealing: and sealing with a sealing agent.
Preferably, the xylene is used for soaking in step i) for more than two times, and the soaking time is preferably 12-16 minutes, and more preferably 13-15 minutes.
Preferably, the soaking time in the step i) with absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol and water is 2-5 minutes, and more preferably 2-3 minutes.
Preferably, the time for soaking and dyeing with the hematoxylin dyeing solution in the step ii) is 10-18 minutes, and more preferably 12-16 minutes.
Preferably, after the hematoxylin staining solution is used for soaking and staining in the step ii), the differentiation solution is used for differentiation after the hematoxylin staining solution is washed for 1-2 minutes by running water.
Preferably, the time for soaking differentiation with the differentiation solution in step ii) is less than 10 seconds, so that the slices fade to a bluish red color.
Preferably, the time of flushing with running water in the step ii) is 5-15 minutes, so that the tissue is bright blue or sky blue.
Preferably, the soaking time in step ii) with 80% ethanol is less than 10 seconds.
Preferably, the time for soaking and dyeing with eosin dye solution in the step ii) is 0.5-2 minutes.
Preferably, the time for soaking and dehydrating in 80% ethanol in step iii) is less than 1 minute.
Preferably, the soaking and dehydrating in the step iii) with 95% ethanol are performed for more than two times, and the soaking time is 1-2 minutes.
Preferably, the soaking and dehydrating in the ethanol in the step iii) are performed for more than two times, and the soaking time is 2-4 minutes.
Preferably, the number of times of soaking in xylene for transparency in the step iv) is more than two, and the soaking time is 2-4 minutes.
In the description of the present invention, the percentage concentration of the ethanol solution refers to the volume percentage of the absolute ethanol in the ethanol solution.
The tissue section is subjected to interstitial staining by adopting hematoxylin-eosin (HE), the hematoxylin-eosin staining solution is different in affinity and staining property to basic dye (hematoxylin) and acidic dye (eosin) mainly according to different components of the tissue or the cells, so after the cell or the tissue section is stained by the hematoxylin-eosin staining solution, cell nuclei and the like mainly composed of acidic substances are stained into bright bluish purple by the hematoxylin, and various different components of cytoplasm, muscle fiber, collagen fiber and the like containing the basic substances present red with different depths. For example, muscle fiber is deep red, red blood cells are orange red, calcium is deep blue, and mucin is gray blue. However, the color differences are very slight, and the accurate identification and quantification are difficult by naked eyes, so that the accurate quantification of the tumor intercellular substance can be realized with the help of a digital pathological scanning analysis system, and the ratio of the tumor cells to the interstitial part in the tumor tissue can be calculated, so that the method can be used for tumor prognosis evaluation and therapeutic drug screening.
The color names are only used for illustrating color differences, and are not absolute colors, in practical situations, the result may have subjective or objective errors, but the technical principle of distinguishing components of cell nucleus, cytoplasm and intercellular substance by combining morphologies through color differences is not influenced.
Furthermore, the digital pathological scanning analysis system calculates the ratio of the intercellular substance in the tumor region tissue through the color difference and morphological difference of the stained intercellular substance and tumor cells, and finally evaluates the prognosis of the tumor patient and screens a proper therapeutic drug through the numerical value of the tumor-interstitial ratio.
Further, the invention provides a method for carrying out tumor interstitial ratio digital pathology scanning analysis by using the kit, which comprises the following steps:
A) hematoxylin-eosin staining of tumor sections;
B) carrying out digital pathological scanning on the slices;
C) selecting a tumor area;
D) and (5) processing and analyzing the images, and calculating to obtain the tumor stroma ratio.
Preferably, the magnification of the digital pathological scanning of the slice in the step B) is 40 times, and the resolution is 0.15-0.30 mu m per pixel.
In some embodiments of the invention, the selecting of the tumor area in step C) is performed manually. In other embodiments of the present invention, the selection is automatically selected by the analysis system. In still other embodiments of the present invention, the selection is an automated selection in combination with a human-assisted selection for the analysis system.
Preferably, the tumor area selected in step C) should correspond to: tumor cells were present at all borders and surrounding stromal tissue without tumor cells was not selected.
Preferably, the image processing in step D) includes: white balance correction, intensity standardization, color normalization, color channel extraction, filtering and threshold processing of the hematoxylin-stained image and the hematoxylin-eosin-stained image separately, and combination of processing results.
Further, the step D) is a tumor-to-stroma ratio analysis step, which is implemented by an established tumor-to-stroma ratio analysis model in a digital pathology scanning analysis system, and the establishment method of the tumor-to-stroma ratio analysis model is as follows:
a) selecting a tumor region in the image, then carrying out tumor and stroma labeling on the obtained tumor region image, and forming a training set by the labeled image;
b) and training the selected convolutional neural network by using the training set to form a tumor-to-mesenchymal ratio analysis model.
In some embodiments of the invention, the labeling of the tumor and stroma in step a) is an artificial labeling; in other embodiments of the present invention, the label is an automated label for the analysis system; in still other embodiments of the present invention, the annotation incorporates human-assisted annotation for automated annotation of the analysis system. Wherein, the automatic labeling of the analysis system is also labeled by a tumor interstitial ratio analysis model formed by a trained convolutional neural network.
In a preferred embodiment of the invention, the tumor region is automatically labeled by a model formed by a trained convolutional neural network, then the tumor region is manually checked and corrected, and the image of the labeled tumor region after being checked and corrected is added into a training set again to retrain the model so as to enable the model to be perfect. The method is also suitable for the automatic selection of the analysis system for selecting the tumor area.
Further, the tumor-to-stroma ratio refers to a ratio of the stromal components in the selected tumor region.
Preferably, the relationship between the calculated tumor-to-stroma ratio and the prognostic assessment is: the low interstitial ratio is obtained when the interstitial ratio is 50% or less, the high interstitial ratio is obtained when the interstitial ratio is 50% or more, and the prognosis is poor when the high interstitial ratio is obtained.
The intercellular substance staining kit for the digital pathology scanning analysis system adopts a hematoxylin-eosin staining system to carry out contrast staining on the intercellular substance, and because the digital pathology scanning analysis system has higher requirements on the accuracy, the contrast and the color uniformity of the staining, the staining solution with a common formula often cannot achieve the ideal effect. The dyeing effect of the traditional hematoxylin-eosin dyeing solution is greatly influenced by temperature, and the dyeing solution disclosed by the invention has a stable effect within a temperature range of 10-30 ℃ and almost has no color difference.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is an image of a stained section tissue in which the nucleus is stained blue or black, the cytoplasm is stained pink or purple pink, the muscle fiber is stained deep red, the calcium is stained deep blue, and the mucin is stained gray blue;
FIG. 2 is three images during analysis shown with a low-to-medium ratio sample, A being a stained image of a tumor tissue region before analysis, B being a processed image of the tumor tissue region during analysis, and C being a resultant image of the tumor tissue region after analysis;
FIG. 3 is three images during analysis shown with a high-to-medium ratio sample, A being a stained image of a tumor tissue region before analysis, B being a processed image of the tumor tissue region during analysis, and C being a resultant image of the tumor tissue region after analysis;
FIG. 4 is a schematic flow chart of an analysis method of the digital pathology scan analysis system.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the present invention.
Example 1
The intercellular substance staining kit for the digital pathological scanning analysis system comprises hematoxylin staining solution, eosin staining solution and differentiation solution, wherein each liter of hematoxylin staining solution contains: 1.9 g of hematoxylin, 18 g of aluminum potassium sulfate, 3.5 g of sodium iodate, 200 mL of glycerol, 5 mL of ethanol and the balance of water; each liter of eosin dye solution contains: eosin Y7.5 g, ethanol 900 mL, and the balance water; each liter of differentiation solution contains the following components: 650 mL of ethanol, 8 mmol of HCl and the balance of water. The kit is stored at 10-30 ℃ and has an effective period of 18 months.
The preparation method of the hematoxylin staining solution comprises the following steps:
1) preparing a hematoxylin alcohol solution: dissolving 1.9 g of hematoxylin in 5 mL of absolute ethyl alcohol;
2) preparing a mixed aqueous solution: fully dissolving 18 g of aluminum potassium sulfate by using distilled water, and then adding 200 mL of glycerol and 3.5 g of sodium iodate;
3) adding the hematoxylin alcohol solution into the mixed aqueous solution, adding water to 1000 mL, and uniformly oscillating to obtain the hematoxylin staining solution.
The preparation method of the eosin dye solution comprises the following steps:
eosin stain was prepared by dissolving 7.5 g eosin Y directly in 1000 mL of 90% ethanol.
The preparation method of the differentiation solution comprises the following steps:
0.67 mL of concentrated hydrochloric acid (12M in concentration) was added dropwise to 1000 mL of 65% ethanol.
Example 2
The intercellular substance staining kit for the digital pathological scanning analysis system comprises hematoxylin staining solution, eosin staining solution and differentiation solution, wherein each liter of hematoxylin staining solution contains: 4.0 g of hematoxylin, 25 g of aluminum potassium sulfate, 4.5 g of sodium iodate, 220 mL of glycerol, 15 mL of ethanol and the balance of water; each liter of eosin dye solution contains: eosin Y12.5 g, ethanol 950 mL, and the balance water; each liter of differentiation solution contains the following components: 700 mL of ethanol, 17 mmol of HCl and the balance of water. The kit is stored at 10-30 ℃ and has an effective period of 18 months.
The preparation method of the hematoxylin staining solution comprises the following steps:
1) preparing a hematoxylin alcohol solution: dissolving 4.0 g of hematoxylin in 15 mL of absolute ethyl alcohol;
2) preparing a mixed aqueous solution: fully dissolving 25 g of aluminum potassium sulfate by using distilled water, properly heating to assist in dissolving, wherein the heating temperature is 80-90 ℃, then adding 4.5 g of sodium iodate, and uniformly mixing;
3) adding the hematoxylin alcohol solution into the mixed aqueous solution, then adding 220 mL of glycerol, adding water to 1000 mL, and uniformly shaking to obtain the hematoxylin staining solution.
The preparation method of the eosin dye solution comprises the following steps:
eosin stain was prepared by dissolving 12.5 g eosin Y directly in 1000 mL of 95% ethanol.
The preparation method of the differentiation solution comprises the following steps:
1.42 mL of concentrated hydrochloric acid (12M in concentration) was added dropwise to 1000 mL of 70% ethanol.
Example 3
The intercellular substance staining kit for the digital pathological scanning analysis system comprises hematoxylin staining solution, eosin staining solution and differentiation solution, wherein each liter of hematoxylin staining solution contains: 2.2 g of hematoxylin, 15 g of aluminum potassium sulfate, 3.8 g of sodium iodate, 180 mL of glycerol, 10 mL of ethanol and the balance of water; each liter of eosin dye solution contains: eosin Y8.0 g, ethanol 980 mL, the rest is water; each liter of differentiation solution contains the following components: 750 mL of ethanol, 10 mmol of HCl and the balance of water. The kit is stored at 10-30 ℃ and has an effective period of 18 months.
The preparation method of the hematoxylin staining solution comprises the following steps:
1) preparing a hematoxylin alcohol solution: dissolving 1.1 g of hematoxylin in 5 mL of absolute ethyl alcohol;
2) preparing a mixed aqueous solution: fully dissolving 7.5 g of aluminum potassium sulfate by using distilled water, and then adding 90 mL of glycerol and 1.9 g of sodium iodate;
3) adding the hematoxylin alcohol solution into the mixed aqueous solution, adding a drop of glacial acetic acid, then adding water to 500 mL, and uniformly shaking to obtain the hematoxylin staining solution.
The preparation method of the eosin dye solution comprises the following steps:
eosin stain was prepared by dissolving 4.0 g eosin Y directly in 500 mL of 98% ethanol and adding two drops of glacial acetic acid.
The preparation method of the differentiation solution comprises the following steps:
0.42 mL of concentrated hydrochloric acid (12M in concentration) was added dropwise to 500 mL of 75% ethanol.
Example 4
The intercellular substance staining kit for the digital pathological scanning analysis system comprises hematoxylin staining solution, eosin staining solution and differentiation solution, wherein each liter of hematoxylin staining solution contains: 3.6 g of hematoxylin, 22 g of aluminum potassium sulfate, 4.2 g of sodium iodate, 200 mL of glycerol, 12 mL of ethanol and the balance of water; each liter of eosin dye solution contains: eosin Y12.0 g, ethanol 980 mL, the rest is water; each liter of differentiation solution contains the following components: 700 mL of ethanol, 14 mmol of HCl and the balance of water. The kit is stored at 10-30 ℃ and has an effective period of 18 months.
The preparation method of the hematoxylin staining solution comprises the following steps:
1) preparing a hematoxylin alcohol solution: dissolving 1.8 g of hematoxylin in 6 mL of absolute ethyl alcohol;
2) preparing a mixed aqueous solution: fully dissolving 11 g of aluminum potassium sulfate by using distilled water, properly heating to assist in dissolving, wherein the heating temperature is 80-90 ℃, then adding 2.1 g of sodium iodate, and uniformly mixing;
3) adding the hematoxylin alcohol solution into the mixed aqueous solution, then adding 100 mL of glycerol and one drop of glacial acetic acid, adding water to 500 mL, and uniformly shaking to obtain the hematoxylin staining solution.
The preparation method of the eosin dye solution comprises the following steps:
eosin stain was prepared by dissolving 6.0 g of eosin Y directly in 500 mL of 98% ethanol and a drop of glacial acetic acid was added.
The preparation method of the differentiation solution comprises the following steps:
0.58 mL of concentrated hydrochloric acid (12M in concentration) was added dropwise to 500 mL of 70% ethanol.
EXAMPLE 5 preparation of Paraffin sections
1.1 immersing the tumor tissue mass removed by the operation in a fixing solution to denature and coagulate the proteins of the tissue and cells, thereby preventing autolysis or bacterial decomposition after cell death and maintaining the original morphological structure of the cells.
1.2 soaking in low-concentration to high-concentration alcohol for dehydration, gradually removing water from the tissue block, and then placing the tissue block in xylene for transparency.
1.3, placing the transparent tissue block into melted paraffin, and placing the tissue block into a paraffin dissolving box for heat preservation. Embedding after the paraffin is completely immersed into the tissue block: and putting the tissue block soaked with the paraffin into the melted paraffin, and cooling and solidifying the tissue block into a block.
1.4 the embedded wax block is fixed on a slicer, cut into slices with the thickness of 4-6 mu m, put into hot water for ironing, then pasted on a glass slide, and put into a thermostat for drying or baked on a heating device.
Example 6 interstitial hematoxylin-eosin staining procedure and results
Using the intercellular substance staining kit for digital pathology scanning analysis system prepared in examples 1-4, serial tumor pathological sections were stained at 10 deg.C, 25 deg.C, and 30 deg.C, respectively, as follows.
Dewaxing
1.1 the dried or roasted slices were placed in xylene I (1 jar dewaxed) and xylene II (2 jar dewaxed) in sequence for 15 minutes to dewax. The dewaxing time is determined by whether the paraffin is completely dissolved or not, the time can be prolonged when the temperature is low, and the time can be properly shortened when the temperature is high.
1.2 sequentially putting into absolute ethyl alcohol, 95 percent ethyl alcohol and 80 percent ethyl alcohol for 2 minutes respectively.
1.3 putting the mixture into water for 2-3 minutes, and washing off ethanol.
Secondly, dyeing
2.1 putting the raw materials into hematoxylin for dip dyeing for 15 minutes, and flushing the raw materials for 1 to 2 minutes by running water.
2.2 differentiate in differentiation medium for 5 seconds, fade the sections to light blue-red. The differentiation has the effect of removing blue cytoplasm and making cell nucleus more clear and fresh. When differentiation is insufficient, the cytoplasm is blue, and the nucleus is over-stained; when the differentiation is excessive, the nucleus is too faint and difficult to be identified, and the hematoxylin staining solution can be returned for a certain time.
2.3 flushing with running water for 10 minutes, the tissue appears bright blue or sky blue (bluish).
2.4 the glass slide is firstly put into 80 percent ethanol for soaking for 5 seconds and then is soaked in eosin solution for 1 minute without water washing.
Thirdly, dehydration
Dehydration in 3.180% ethanol for 30 s, and re-dyeing in eosin solution if the color fading in ethanol is rapid.
Dehydration in 3.295% ethanol I, II for 1 min and 2 min, respectively.
3.3 Anhydrous ethanol I, II each 2 minutes.
Fourthly, transparent
4.1 clear in xylene I, xylene II for 2 minutes each.
Fifthly, sealing and fixing
5.1 blocking with Eukit.
Sixthly, scanning analysis
Placing the stained tissue slide glass under a microscope of a digital pathological scanning analysis system, and scanning by using a multiplying power of 40 times, wherein the resolution is 0.23 mu m per pixel; selecting a tumor area by a pathologist, selecting an area with tumor cells at all boundaries, and not selecting surrounding stromal tissue without the tumor cells; then the analysis system carries out image processing and interstitial ratio analysis, and the tumor interstitial ratio is calculated.
Seven, result in
FIG. 1 shows an image of stained tissue sections in which the nuclei are stained blue or black, the cytoplasm is stained pink or purple pink, the muscle fibers are stained deep red, the calcium is stained deep blue, and the mucin is stained gray blue.
Fig. 2 and 3 show three images during the interstitial ratio analysis, a being a stained image of the tumor tissue region before analysis, B being a processed image of the tumor tissue region during analysis, and C being a resultant image of the tumor tissue region after analysis. The image a shows the aforementioned various tissue components with subtle color differences, the image B is a classification map presented after processing by analysis software, wherein dark gray (primary red) is tumor cells, light gray (primary green) is stroma, black (primary blue) is blank, and the image C is a visual image obtained after the color of the stroma is lightened to form a larger color difference with the tumor cells after the analysis is completed. Finally, the tumor-to-stroma ratio is automatically calculated by software according to the tumor-to-stroma classified above, and as can be seen from fig. 2, the image is a low-stroma ratio image with a stroma ratio value of 18%, and as can be seen from fig. 3, the image is a high-stroma ratio image with a stroma ratio value of 65%.
Serial sections of the same sample were stained at 10 ℃, 25 ℃ and 30 ℃ using the kits of examples 1-4, respectively, and then subjected to interstitial ratio analysis using a digital pathology scanning analysis system, with the results: the calculated interstitial ratio values of the same kit at three temperatures and different kits at the same temperature are the same (the ratio of the interstitial to the area of the selected tumor region is reserved with two decimal places), and the kit disclosed by the invention has a stable result within the range of 10-30 ℃.
Example 7 analysis procedure for digital pathological Scan analysis System
The principle flow of the scanning analysis method described in this embodiment is shown in fig. 4, and includes the steps of: hematoxylin-eosin staining of tumor sections; carrying out digital pathological scanning on the slices; selecting a tumor area; and (5) processing and analyzing the images, and calculating to obtain the tumor stroma ratio.
Wherein, the tumor region selection is completed in the scanned image by the pathology household software, the region with the tumor cells at all the boundaries is selected, and the surrounding stroma tissue without the tumor cells is not selected. In some cases, the analysis can be performed automatically by an analysis system, and the selected convolutional neural network can be trained to select the tumor region when the analysis system is set up.
The image processing and analysis are completed by an established tumor interstitial ratio analysis model, and the model establishment method comprises the following steps: selecting a tumor region in the image, then carrying out tumor and stroma labeling on the obtained tumor region image, and forming a training set by the labeled image; and training the selected convolutional neural network by using the training set to form a tumor-to-mesenchymal ratio analysis model. In the initial stage of model establishment, a pathologist selects a tumor region and labels tumors and stroma, the labeled images form a training set to train the selected convolutional neural network, when the initial convolutional neural network is trained (for example, the training set is 50 images), the model which is trained primarily is used to label (or can comprise selection) other images, then the pathologist checks and corrects the images, and then the images are added into the training set to retrain the initial model (for example, 20 images are added, and the training set is changed into 70 images), and the process is repeated continuously to enable the model to be perfect. Finally, a mature tumor interstitial ratio analysis model is obtained.
The calculated relationship between the tumor stroma ratio and the prognosis evaluation is: the low interstitial ratio is obtained when the interstitial ratio is 50% or less, the high interstitial ratio is obtained when the interstitial ratio is 50% or more, and the prognosis is poor when the high interstitial ratio is obtained. Compared with the original manual calculation result, the calculated tumor stroma ratio numerical value has the advantages of obviously reduced error, higher accuracy and greatly improved speed. The working efficiency is greatly improved for the hospital pathology department with hundreds of pathological sections to be diagnosed every day.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A intercellular substance staining kit for digital pathological scanning analysis system is characterized by comprising hematoxylin staining solution, eosin staining solution and differentiation solution; wherein the content of the first and second substances,
each liter of hematoxylin staining solution contains the following components: 1.9-4.0 g of hematoxylin, 15-25 g of aluminum potassium sulfate, 3.5-4.5 g of sodium iodate, 180-220 mL of glycerol, 5-15 mL of ethanol and the balance of water;
every liter of eosin dye solution contains the following components: 7.5-12.5 g of eosin Y, 900-980 mL of ethanol and the balance of water;
each liter of differentiation solution contains the following components: 650-800 mL of ethanol, 8-17 mmol of HCl and the balance of water.
2. The intercellular substance staining kit for digital pathology scanning analysis system according to claim 1, wherein the kit is preserved at 10-30 ℃ for 6-24 months.
3. The intercellular staining kit for digital pathology scan analysis system of claim 1, further comprising a fixative.
4. The intercellular substance staining kit for digital pathology scan analysis system of claim 1, further comprising any one or more of absolute ethanol, 95% ethanol, 80% ethanol, xylene.
5. The intercellular substance staining kit for digital pathology scanning analysis system according to claim 1, wherein the preparation method of said hematoxylin staining solution comprises the steps of:
1) dissolving hematoxylin with anhydrous ethanol;
2) dissolving aluminum potassium sulfate in water;
3) mixing the ethanol solution of the hematoxylin obtained in the step 1) with the aqueous solution of the potassium aluminum sulfate obtained in the step 2) and the sodium iodate;
4) adding glycerol into the aluminum potassium sulfate aqueous solution obtained in the step 2) or the mixed solution obtained in the step 3).
6. The intercellular substance staining kit for digital pathology scanning analysis system according to claim 1, wherein said eosin staining solution is directly prepared by dissolving eosin Y in an ethanol solution.
7. The intercellular substance staining kit for digital pathology scan analysis system according to claim 1, wherein the differentiation solution is prepared by dropping concentrated hydrochloric acid into ethanol solution in calculated amount.
8. Method for interstitial staining of pathological sections for digital pathological scanning analysis systems with the staining kit according to any of claims 1 to 7, characterized in that it comprises the steps of:
i) dewaxing: soaking and dewaxing by using dimethylbenzene, and then sequentially soaking and washing by using absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol and water;
ii) dyeing: sequentially soaking and dyeing with hematoxylin dye solution, soaking and differentiating with differentiation solution, washing with running water, soaking with 80% ethanol, and soaking and dyeing with eosin dye solution;
iii) dehydration: soaking in 80% ethanol, 95% ethanol, and anhydrous ethanol sequentially for dehydration;
iv) transparency: soaking in xylene to make it transparent;
v) sealing: and sealing with a sealing agent.
9. Method for tumor interstitial ratio digital pathology scan analysis of tumor sections with the staining kit according to any of claims 1 to 7, characterized in that it comprises the steps of:
A) hematoxylin-eosin staining of tumor sections;
B) carrying out digital pathological scanning on the slices;
C) selecting a tumor area;
D) and (5) processing and analyzing the images, and calculating to obtain the tumor stroma ratio.
10. The method for analyzing tumor-to-stroma ratio digital pathological scan of claim 9, wherein the analysis is performed by an established tumor-to-stroma ratio analysis model in the digital pathological scan analysis system, and the tumor-to-stroma ratio analysis model is established by:
a) selecting a tumor region in the image, then carrying out tumor and stroma labeling on the obtained tumor region image, and forming a training set by the labeled image;
b) and training the selected convolutional neural network by using the training set to form a tumor-to-mesenchymal ratio analysis model.
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