A hematoxylin-eosin staining solution, and a preparation method and application thereof

Abstract

The present application relates to a kind of hematoxylin-eosin staining solution and its preparation method and application, belong to biotechnology technical field.The hematoxylin-eosin staining solution of the present application includes hematoxylin system and eosin system, hematoxylin system includes hematoxylin, anhydrous ethanol, potassium aluminum sulfate, potassium iodate, potassium permanganate, citric acid, polyethylene glycol and water, and eosin system includes eosin Y, triethanolamine and tween 80.The hematoxylin-eosin staining solution of the present application is compatible with artificial dyeing operation and full-automatic pathological high-definition dyeing equipment, with the advantages of high-definition, constant dyeing stability, no batch color difference, long storage period and wide adaptability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a hematoxylin-eosin staining solution, its preparation method, and its application. Background Technology

[0002] Hematoxylin-eosin (HE) staining is a core foundational technique for pathological diagnosis. More than 80% of clinical pathological diagnostic reports are completed based on HE-stained slides. By staining the basophilic structures of cell nuclei with hematoxylin and the eosin staining of the eosinophilic structures of cytoplasm and intercellular matrix with eosin, the structure of tissue cells can be visualized and differentiated. It is the core basis for pathological screening, disease classification, and lesion determination.

[0003] Currently, conventional HE staining solutions generally suffer from several technical defects, including: a single staining component, insufficient clarity after staining, blurred cell boundaries, low recognition of fine tissue structures, inability to clearly distinguish minute lesions, and a high risk of missed or misdiagnosed lesions; significant batch-to-batch color variations leading to poor staining stability, uneven staining depth in different areas of the same section, poor compatibility between manual and machine staining, and inability to meet the standardized batch staining requirements of fully automated staining equipment; poor color durability, with stained sections fading, turning white, and darkening quickly, failing to meet the requirements for long-term archiving and traceability of clinical sections; insufficient reagent storage stability, with long-term storage prone to solute precipitation, liquid stratification, and oxidation of active ingredients, resulting in short reagent shelf life and increased pathological testing costs and operational errors due to frequent reagent replacements; and weak anti-interference ability, easily affected by residual tissue impurities, environmental temperature and humidity, and staining time, resulting in poor staining repeatability. These numerous defects seriously affect the accuracy of pathological diagnosis and the effectiveness of section archiving.

[0004] Most existing modified HE staining solutions only optimize a single performance aspect and cannot simultaneously achieve the comprehensive effects of high-definition color development, constant staining stability, long-term storage, and standardized batch staining. Furthermore, they have poor compatibility and are difficult to match the usage requirements and urgent needs of high-end clinical pathology high-definition staining systems. Summary of the Invention

[0005] The purpose of this invention is to provide a hematoxylin-eosin staining solution, its preparation method, and its application, in order to solve the problem that existing HE staining solutions cannot simultaneously achieve the comprehensive effects of high-definition color development, constant staining stability, strong adaptability, long-term storage, and standardized batch staining.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a hematoxylin-eosin staining solution, comprising a hematoxylin system and an eosin system;

[0008] The hematoxylin system comprises the following components in parts by weight: 1.0–1.5 parts hematoxylin, 8–12 parts anhydrous ethanol, 15–20 parts potassium aluminum sulfate, 0.15–0.25 parts potassium iodate, 0.05–0.1 parts potassium permanganate, 0.2–0.4 parts citric acid, 0.3–0.6 parts polyethylene glycol, and 65.15–75.3 parts water;

[0009] The eosin system comprises the following components in parts by mass: eosin Y 0.8–1.2 parts, triethanolamine 0.5–1.0 parts, Tween 80 0.1–0.3 parts, and water 90–100 parts.

[0010] The present invention also provides a method for preparing the hematoxylin-eosin staining solution, comprising the following steps:

[0011] (1) Stir water for 5-10 minutes, then add potassium aluminum sulfate and stir for 10-20 minutes to obtain the base solution;

[0012] (2) Dissolve hematoxylin in anhydrous ethanol to obtain a solution, mix the solution with the base liquid and stir for 20-30 minutes to obtain a mixture;

[0013] (3) Mix the mixture with potassium iodate, potassium permanganate and citric acid and stir for 20-30 minutes to obtain an oxidation color development system;

[0014] (4) Mix the oxidation color development system with polyethylene glycol, stir for 20-40 min, let stand for 24-36 h and then filter to obtain the hematoxylin system;

[0015] (5) Mix eosin Y and water and stir for 10-15 minutes, then add triethanolamine and Tween 80 and stir for 15-20 minutes to obtain a premixed solution;

[0016] (6) After the premixed solution is allowed to stand for 6 to 18 hours, it is filtered to obtain the eosin system.

[0017] Preferably, the stirring speed in step (1) is 250 to 350 rpm.

[0018] Preferably, the mixing time in step (2) is 8 to 15 minutes;

[0019] The stirring speed is 250-350 rpm.

[0020] Preferably, the stirring speed in step (3) is 350 to 500 rpm.

[0021] Preferably, the stirring speed in step (4) is 350-500 rpm;

[0022] The filtration method is filtration using a 0.22μm sterile microporous membrane.

[0023] Preferably, the stirring speed in step (5) is 250 to 350 rpm.

[0024] Preferably, the filtration method in step (6) is filtration using a 0.45 μm sterile microporous membrane.

[0025] The present invention also provides the application of the hematoxylin-eosin staining solution in cell and / or tissue staining.

[0026] The present invention also provides the application of the hematoxylin-eosin staining solution prepared by the above preparation method in cell and / or tissue staining.

[0027] The present invention has the following technical effects and advantages:

[0028] The hematoxylin-eosin staining solution of the present invention adopts a dual-system formula, which can accurately adapt to the staining rhythm of fully automated high-definition pathological staining equipment, without the need for repeated manual solution adjustment and calibration, and has extremely high batch staining consistency. At the same time, it is compatible with manual staining operations and has the advantages of high-definition color development, stable constant staining, no batch color difference, long storage period, and wide compatibility. It can meet the application and needs of accurate pathological diagnosis, standardized batch staining and detection of tissue and cell sections, long-term archiving and traceability, etc.

[0029] The hematoxylin-eosin staining solution of this invention, through optimized dual-system formulation, complex oxidation control, pH stabilization, and synergistic improvement of fixation and staining, exhibits significant advantages over commercially available conventional HE staining solutions in six core performance aspects: clarity of microscopic color development, staining uniformity, batch consistency, long-term storage stability, section fading resistance, and bidirectional equipment compatibility. The reagent eliminates batch-to-batch color differences and long-term precipitation failure issues, resulting in clearly defined stained sections and high lesion identification. It can simultaneously meet the needs of precise manual staining and standardized batch staining using fully automated equipment, perfectly adapting to core application scenarios such as precise clinical pathology diagnosis, batch sample testing, and long-term archiving and traceability of sections. It solves several industry pain points of traditional HE staining solutions and possesses extremely high clinical promotion and industrial application value.

[0030] This invention combines potassium iodate and potassium permanganate as a composite oxidant in the hematoxylin system, precisely controlling the degree of hematoxylin oxidation. This ensures deep and clear staining of cell nuclei with clear boundaries, while avoiding excessive oxidation that leads to dark staining and a cluttered background, thus completely solving the batch-to-batch color difference defects of traditional staining solutions. Citric acid is added to adjust the pH of the system, eliminating acid-base interference in staining and preventing section color differences. Polyethylene glycol is added as a stabilizer to effectively encapsulate the active ingredients of the staining solution, inhibiting solute precipitation and oxidation failure, solving the problem of layering and precipitation that occurs during long-term storage of traditional HE staining solutions, and significantly extending the shelf life of the reagents.

[0031] This invention adds triethanolamine as a fixing agent to the eosin system, which can form stable bonds with cytoplasmic chromatids, greatly improving staining adhesion and color durability, avoiding fading and discoloration of sections, and achieving long-term constant staining effect; adding Tween 80 as a auxochromating agent improves the uniformity of staining solution penetration and eliminates the problem of uneven staining depth in some areas. Attached Figure Description

[0032] Figure 1 Microscopic images of pathological tissue sections from the experimental group;

[0033] Figure 2 Microscopic images of pathological tissue sections from the control group. Detailed Implementation

[0034] This invention provides a hematoxylin-eosin staining solution, comprising a hematoxylin system and an eosin system;

[0035] The hematoxylin system comprises the following components in parts by weight: 1.0-1.5 parts hematoxylin, preferably 1.2 parts; 8-12 parts anhydrous ethanol, preferably 10 parts; 15-20 parts potassium aluminum sulfate, preferably 18 parts; 0.15-0.25 parts potassium iodate, preferably 0.2 parts; 0.05-0.1 parts potassium permanganate, preferably 0.08 parts; 0.2-0.4 parts citric acid, preferably 0.3 parts; 0.3-0.6 parts polyethylene glycol, preferably 0.45 parts; and 65.15-75.3 parts water, preferably 69.77 parts.

[0036] The eosin system comprises the following components in parts by mass: eosin Y 0.8-1.2 parts, preferably 1.0 parts; triethanolamine 0.5-1.0 parts, preferably 0.7 parts; Tween 80 0.1-0.3 parts, preferably 0.2 parts; and water 90-100 parts, preferably 97.1 parts.

[0037] The present invention also provides a method for preparing the hematoxylin-eosin staining solution, comprising the following steps:

[0038] (1) Stir water for 5 to 10 minutes, preferably 5 minutes, then add potassium aluminum sulfate and stir for 10 to 20 minutes, preferably 15 minutes, to obtain the base solution;

[0039] (2) Dissolve hematoxylin in anhydrous ethanol to obtain a solution, mix the solution with the base liquid and stir for 20-30 min, preferably 20 min, to obtain a mixture;

[0040] (3) Mix the mixture with potassium iodate, potassium permanganate and citric acid and stir for 20-30 min, preferably 25 min, to obtain an oxidation color development system;

[0041] (4) Mix the oxidative color development system with polyethylene glycol, stir for 20-40 min, preferably 30 min, let stand for 24-36 h, preferably 24 h, and then filter to obtain the hematoxylin system.

[0042] (5) Mix eosin Y and water and stir for 10-15 min, preferably 15 min, then add triethanolamine and Tween 80 and stir for 15-20 min, preferably 20 min, to obtain a premixed solution;

[0043] (6) Let the premixed solution stand for 6 to 18 hours, preferably 12 hours, and then filter to obtain the eosin system.

[0044] (6) Let the premixed solution stand for 6 to 18 hours, preferably 12 hours, and then filter to obtain the eosin system.

[0045] In this invention, the mixing time in step (2) is 8 to 15 minutes, preferably 10 minutes;

[0046] The mixing method is slow drip infusion;

[0047] The stirring speed is 250-350 rpm, preferably 300 rpm.

[0048] In this invention, the stirring speed in step (3) is 350-500 rpm, preferably 400 rpm.

[0049] In this invention, the stirring speed in step (4) is 350-500 rpm, preferably 400 rpm;

[0050] The filtration method is filtration using a 0.22μm sterile microporous membrane.

[0051] In this invention, the stirring speed in step (5) is 250-350 rpm, preferably 300 rpm.

[0052] In this invention, the filtration method in step (6) is filtration using a 0.45μm sterile microporous membrane;

[0053] The method for allowing the material to stand is to let it stand at room temperature and away from light.

[0054] The present invention also provides the application of the hematoxylin-eosin staining solution in cell and / or tissue staining.

[0055] The present invention also provides the application of the hematoxylin-eosin staining solution prepared by the above preparation method in cell and / or tissue staining.

[0056] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0057] The test materials of this invention include paraffin pathological sections of human colon polyps, breast hyperplasia, and lung tissue, with 60 parallel sections of each tissue, all without damage, detachment, or tissue defects, and all subjected to routine dewaxing and water pretreatment, and stored at Ningbo Cha Microbial Technology Co., Ltd.; the high-end commercial HE staining kit was purchased from Wuhan Saiwei Biotechnology Co., Ltd.

[0058] Example 1: Preparation of the hematoxylin system

[0059] At 25℃, 69.77g of water was added to the reactor and stirred at 300rpm for 5min until the water volume stabilized. Then, 18g of potassium aluminum sulfate was slowly added and stirred for another 15min until completely dissolved to obtain the base solution. 1.2g of hematoxylin was dissolved in 10g of anhydrous ethanol to obtain a solution. This solution was slowly added dropwise to the base solution over 10min, and stirred at 300rpm for 20min to obtain a mixture. 0.2g of potassium iodate, 0.08g of potassium permanganate, and 0.3g of citric acid were added sequentially to the mixture, and stirred at 400rpm for 25min to obtain the oxidation colorimetric system. 0.45g of polyethylene glycol was added to the oxidation colorimetric system, and stirred at 400rpm for 30min to mix thoroughly. The mixture was then allowed to stand in the dark for 24h to mature, and filtered through a 0.22μm sterile microporous membrane to obtain the hematoxylin system.

[0060] Example 2: Preparation of the eosin system

[0061] At 25°C, 97.1g of water was added to the mixing tank, and then 1.0g of eosin Y was slowly added and stirred continuously for 15min until completely dissolved. Then, 0.7g of triethanolamine and 0.2g of Tween 80 were added in sequence and stirred at 300rpm for 20min until uniformly mixed to obtain a premixed solution. The premixed solution was allowed to stand in the dark for 12h to mature, and then filtered through a 0.45μm sterile microporous membrane to obtain the eosin system.

[0062] Experimental Example 1: Staining of Histopathological Sections

[0063] The aim is to compare the hematoxylin-eosin staining solution prepared in this invention with commercially available conventional HE staining solutions in terms of core performance differences such as tissue color clarity, staining uniformity, batch stability, storage stability, section fading resistance, and equipment compatibility. This will quantitatively verify the technical advantages of the improved formula of this invention and provide data support for standardized staining in clinical pathology, long-term section archiving, and batch application of fully automated equipment.

[0064] Sixty parallel paraffin-embedded pathological sections each of human colonic polyps, breast hyperplasia, and lung tissue were collected. After routine dewaxing and water pretreatment, they were divided into two groups: an experimental group and a control group. The experimental group underwent standardized staining using the hematoxylin system prepared in Example 1 and the eosin system prepared in Example 2, respectively, via manual staining and a standardized fully automated staining machine. The control group used a commercially available high-end HE staining kit, with simultaneous manual and machine-controlled staining. Staining procedures, staining time, differentiation time, and blueing time were standardized to eliminate operational variables. After staining, routine dehydration, clearing, and mounting with neutral resin were performed. Quantitative evaluation was conducted, and the results are as follows: Figures 1-2 As shown in Table 1.

[0065] The quantitative evaluation indicators and scoring criteria adopted a dual evaluation method: a general percentage score for pathological staining (maximum score of 100 points) and instrument quantitative data. The scores were blind-scored by three senior pathology technicians, and the average score was taken. The scoring criteria are as follows:

[0066] 1. Color clarity (30 points): The outline of the cell nucleus is sharp and complete, the nucleoplasm boundary is clear, the fine glands and inflammatory structures are distinguishable, and there is no background noise;

[0067] 2. Staining uniformity (20 points): There is no difference in color depth between individual sections, the staining consistency of sections in the same batch is high, and there are no local omissions or over-staining phenomena;

[0068] 3. Batch stability (20 points): There was no significant difference in staining effect among three consecutive batches of prepared reagents, and the grayscale difference was small;

[0069] 4. Storage stability (15 points): After 6 months of storage at room temperature and away from light, there was no stratification, precipitation, or turbidity, and the staining activity did not decrease.

[0070] 5. Colorfastness (15 points): After being stored in a light-proof, sealed container for 3 months, the slices showed no whitening, darkening, or fading.

[0071] Table 1. Comparison of Quantitative Evaluation Indicators and Scores (Mean Score ± Standard Deviation)

[0072] Color rendering clarity (30 points) 29.12±0.45 22.36±1.21 The cell nuclei in the experimental group were deep and transparent, and the identification of subtle lesion structures was significantly higher. Staining uniformity (20 points) 19.35±0.32 14.58±1.05 The experimental group showed no local color difference, over-dyeing, or missed dyeing, and exhibited excellent uniformity. Batch stability (20 points) 19.56±0.28 13.22±1.36 The average grayscale difference among the three batches of slices in the experimental group was 2.6, while that in the control group was 13.5, indicating a highly significant difference in color between the batches. Storage stability (15 points) 14.68±0.21 9.15±1.52 The experimental group showed no precipitation or stratification after 6 months, while the control group showed solute precipitation and a 42.3% decrease in staining activity after only 2 months. Colorfastness (15 points) 14.82±0.18 8.63±1.64 The experimental group maintained vibrant colors after 3 months of archiving, while the control group showed large areas of whitening and dullness. Total score (100 points) 97.53±1.44 67.94±6.78 The experimental group showed significantly better overall performance than the control group.

[0073] The results showed that, in the experimental group, staining with the hematoxylin-eosin staining solution prepared in this invention resulted in uniform, deep blue-purple cell nuclei with sharp and clear boundaries and extremely high nuclear-cytoplasmic contrast; the cytoplasm and intercellular matrix were bright and uniform pink with distinct layers; glandular structures, microinflammatory lesions, and intercellular fibrous structures were intact and identifiable, with no background staining or nuclear-cytoplasmic co-staining, demonstrating high identification of subtle lesion structures and effectively avoiding missed or misdiagnosed microlesions. In contrast, the control group, stained with a commercially available high-end HE staining kit, showed uneven and faint staining of cell nuclei, with some nuclei having blurred and adhered boundaries; the cytoplasm was cloudy and dark, with obvious non-specific background staining; subtle tissue structures were unclear, with varying staining depths in the same section, making it difficult to accurately distinguish microlesion areas and resulting in poor staining quality stability.

[0074] Experimental Example 2: Batch Stability Determination

[0075] Image grayscale software was used to detect the pathological sections of each tissue after staining in the experimental group and the control group in Experiment Example 1.

[0076] The results showed that when staining with the hematoxylin-eosin staining solution prepared in this invention, the grayscale difference between batches was only 2.6±0.5, with no color difference that could be detected by the naked eye, and the batch consistency was excellent. When staining with commercially available high-end HE staining kits, the grayscale difference between batches was as high as 13.5±1.2, and the batch color difference was obvious, which could not meet the clinical requirements of standardized batch staining with fully automated equipment.

[0077] Experiment Example 3: Storage Stability Determination

[0078] The hematoxylin-eosin staining solution prepared in this invention and a commercially available high-end HE staining kit were stored at room temperature in the dark and sealed for 6 months and continuously measured.

[0079] The results showed that the hematoxylin-eosin staining solution prepared by this invention remained clear and uniform, without layering, precipitation, or turbidity, with an oxidation rate of less than 3% for the effective components. The staining intensity and color development effect were not significantly different from those of the freshly prepared reagent. In contrast, the staining solution of commercially available high-end HE staining kits showed bottom flocculent precipitation and slight liquid layering after 2 months of storage. After 6 months of storage, the oxidation loss rate of the effective components reached 42.3%, and the staining clarity and color intensity decreased significantly, making it unsuitable for long-term storage and use.

[0080] Experiment Example 4: Determination of the fading resistance and archiving performance of sections

[0081] The stained tissue pathological sections of the experimental group and control group in Experiment Example 1 were sealed and archived at room temperature and protected from light for 3 months before observation.

[0082] The results showed that when stained with the hematoxylin-eosin staining solution prepared in this invention, the sections were full and transparent in color, with no attenuation of color saturation, and the contrast between the nucleus and cytoplasm remained clear, fully meeting the requirements for long-term traceability and archiving of clinical pathological sections. When stained with commercially available high-end HE staining kits, the sections generally showed problems such as cytoplasmic whitening, darkening of cell nuclei, and overall fading of color, which greatly reduced the identification of the sections and could not meet the requirements for long-term archiving.

[0083] Experiment Example 5: Equipment Compatibility Test

[0084] A fully automated high-definition pathological staining machine was used to perform batch staining tests on pathological sections of various tissues in the experimental and control groups.

[0085] The results show that the hematoxylin-eosin staining solution prepared in this invention is compatible with the staining rhythm of the equipment, requires no manual calibration or solution adjustment, and can continuously stain 300 sections with uniform and stable staining throughout the process, without abnormal color difference or well blockage. In contrast, commercially available high-end HE staining kits are prone to fluctuations in staining depth and local contamination after batch staining, requiring frequent manual calibration, and have poor compatibility and low work efficiency.

[0086] As can be seen from the above embodiments, the present invention provides a hematoxylin-eosin staining solution, its preparation method, and its application. The hematoxylin-eosin staining solution of the present invention is compatible with both manual staining operations and fully automated high-definition pathological staining equipment. It has the advantages of high-definition color development, stable staining, no batch-to-batch color difference, long storage period, and wide adaptability, and can meet the applications and needs of accurate pathological diagnosis, standardized batch staining and detection of tissues and cell sections, and long-term archiving and traceability.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hematoxylin-eosin staining solution, characterized in that, Including the hematoxylin system and the eosin system; The hematoxylin system comprises the following components in parts by weight: 1.0–1.5 parts hematoxylin, 8–12 parts anhydrous ethanol, 15–20 parts potassium aluminum sulfate, 0.15–0.25 parts potassium iodate, 0.05–0.1 parts potassium permanganate, 0.2–0.4 parts citric acid, 0.3–0.6 parts polyethylene glycol, and 65.15–75.3 parts water; The eosin system comprises the following components in parts by mass: eosin Y 0.8–1.2 parts, triethanolamine 0.5–1.0 parts, Tween 80 0.1–0.3 parts, and water 90–100 parts.

2. The method for preparing the hematoxylin-eosin staining solution according to claim 1, characterized in that, Includes the following steps: (1) Stir water for 5-10 minutes, then add potassium aluminum sulfate and stir for 10-20 minutes to obtain the base solution; (2) Dissolve hematoxylin in anhydrous ethanol to obtain a solution, mix the solution with the base liquid and stir for 20-30 minutes to obtain a mixture; (3) Mix the mixture with potassium iodate, potassium permanganate and citric acid and stir for 20-30 minutes to obtain an oxidation color development system; (4) Mix the oxidation color development system with polyethylene glycol, stir for 20-40 min, let stand for 24-36 h and then filter to obtain the hematoxylin system; (5) Mix eosin Y and water and stir for 10-15 minutes, then add triethanolamine and Tween 80 and stir for 15-20 minutes to obtain a premixed solution; (6) After the premixed solution is allowed to stand for 6 to 18 hours, it is filtered to obtain the eosin system.

3. The preparation method according to claim 2, characterized in that, The stirring speed in step (1) is 250-350 rpm.

4. The preparation method according to claim 3, characterized in that, The mixing time in step (2) is 8 to 15 minutes; The stirring speed is 250-350 rpm.

5. The preparation method according to claim 4, characterized in that, The stirring speed in step (3) is 350-500 rpm.

6. The preparation method according to claim 5, characterized in that, The stirring speed in step (4) is 350-500 rpm; The filtration method is filtration using a 0.22μm sterile microporous membrane.

7. The preparation method according to claim 2, characterized in that, The stirring speed in step (5) is 250-350 rpm.

8. The preparation method according to claim 7, characterized in that, The filtration method described in step (6) is filtration using a 0.45μm sterile microporous membrane.

9. The use of the hematoxylin-eosin staining solution according to claim 1 in cell and / or tissue staining.

10. The application of the hematoxylin-eosin staining solution prepared by the preparation method according to any one of claims 2 to 8 in cell and / or tissue staining.

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