Dyeing solution and dyeing method for urogenital system sample

Abstract

The invention relates to the technical field of cell staining, in particular to a staining solution and a staining method for a urogenital system sample. According to the invention, two dyes with different properties, namely an acid dye and a basic dye, are mixed to prepare a rapid staining solution, and the rapid staining solution can be used for rapidly staining white blood cell nuclei in a sample into blue-green and cytoplasm is not colored through wet sheet staining; cell nuclei of epithelial cells are dyed into dark blue and cytoplasm is dyed into red; live trichomonad is not dyed, and dead trichomonad is dyed into red; therefore, the visible components can be clearly and accurately distinguished. The dyed epithelial cells, leukocytes, trichomonad, candida and the like are clear in structure and obvious in morphological color difference, and AI image analysis is combined, so that the trichomonad identification accuracy can be improved, the instrument trichomonad auditing rate is eliminated, and automatic interpretation is completely realized.

Description

Technical Field

[0001] The invention relates to the technical field of cell staining, in particular to a staining solution and a staining method for urogenital system samples. Background Art

[0002] Due to its unique anatomical structure and physiological functions, the genitourinary system is susceptible to infection by Trichomonas, Candida, and other pathogens, leading to various infectious diseases. These pathogens can enter the genitourinary system through sexual contact, indirect contact, or other pathways, disrupting the normal physiological environment and causing inflammation, infection, and even more serious health problems.

[0003] Currently, urogenital samples are often tested using a combination of morphological and functional testing. Taking vaginal discharge as an example, clinical vaginal discharge testing primarily encompasses four methodologies: saline wet mount microscopy, automated wet mount microscopy, Gram staining, and dry chemical enzymatic testing. The first three fall into the morphological category, while the dry chemical enzymatic method falls into the functional category, and clinical diagnosis primarily relies on morphological results.

[0004] Gram staining microscopy is a routine morphological examination technique for vaginal secretions, but this method has many disadvantages. Not only are the staining steps cumbersome, time-consuming and labor-intensive, but the staining effect is greatly affected by the staining time. Trichomonas flagella are easy to fall off, and the samples on the slide are easily washed away during rinsing, resulting in a lower detection rate of visible components. The overall operation is complicated and is not as efficient as wet mount microscopy.

[0005] Wet mount microscopy is relatively simple. Simply drop the sample onto a glass slide, cover it with a coverslip, and then place it under a microscope for direct observation. One of its advantages is that it can quickly and accurately identify active Trichomonas. However, the contrast of unstained samples prepared by the wet mount method is poor, and the fine cell structure is difficult to clearly distinguish. The operator needs to have a high level of identification ability, especially for microorganisms with similar morphology, which can be easily misjudged. In addition, for Trichomonas samples that have been stored for a long time or have already died, their morphology is similar to that of some white blood cells, and wet mount microscopy cannot accurately distinguish Trichomonas from white blood cells. Among the dyes commonly used for wet mount staining, basic dyes such as safranin, basic fuchsin, and methylene blue have single properties. In addition, the pH value of vaginal secretion samples is not fixed, the degree of cytoplasm binding to the dye varies, and the degree of staining of cells in different cell cycles also varies. As a result, it is impossible to stain all white blood cells, and dead Trichomonas and white blood cells are still difficult to distinguish.

[0006] In the current context of increasingly widespread AI applications, it is particularly important to make it easier for computers to distinguish between different cells. Currently, fully automated wet mount staining microscopy equipment is available on the market. The instrument can automatically mix the sample and staining solution to prepare slides, and automatically focus and take microscopic images of the sample through the microscopy camera system. The software uses AI image recognition algorithms to automatically identify the formed elements in the sample image. However, in application, it was found that although wet mount automated staining microscopy can automatically identify formed elements, some white blood cells are not stained and have a similar morphology to unstained Trichomonas, and the staining of Trichomonas with decreased vitality or death is confused with white blood cells. This can easily cause the instrument's intelligent identification to misjudge or miss detection, and manual review is still required. This not only increases manpower, but also makes it difficult to effectively detect Trichomonas with decreased vitality or death.

[0007] Therefore, further research and development of dyes that can distinguish epithelial cells, leukocytes, Trichomonas and Candida will have positive significance in promoting the detection technology of urogenital system samples to a more accurate, efficient and automated direction. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a staining solution and a staining method for urogenital system samples. More specifically, the technical problem to be solved is to achieve rapid differential staining of formed elements in vaginal secretion samples.

[0009] The dyeing solution provided by the invention comprises acid fuchsin and methyl green, wherein the mass ratio of the acid fuchsin to the methyl green is (20-100):(10-100).

[0010] The present invention adopts acid fuchsin and methyl green as dyes, which can quickly dye different visible elements in the sample into different colors (white blood cell nuclei are dyed into blue-green, epithelial cell cytoplasm is dyed into red and nuclei are dyed into dark blue, living trichomonas are not dyed, dead trichomonas are dyed into red, and molds are not dyed). Each visible element can be accurately identified by morphology and color, and the image after staining is clear under microscopy, and the background is translucent and free of mixed colors.

[0011] In the present invention, the mass ratio of acid fuchsin to methyl green in the dye is (1-2):1. For example, the mass ratio of acid fuchsin to methyl green in the dye is 1:1 or 2:1. In the dye provided by the present invention, acid fuchsin and methyl green may be present alone or in a mixture, and the present invention is not limited thereto. As long as the above mass ratio is met, the implementation falls within the scope of the present invention.

[0012] In the present invention, each component of the dye can be present in the form of a solution or a powder, and the present invention is not limited thereto. If it is present in the form of a solution, the concentration of each component can be the working concentration or 2 to 100 times the working concentration. For example, the concentration of each component can be 2 times, 5 times, 10 times, 20 times, 50 times, or 100 times the working concentration.

[0013] In the present invention, the concentration of acid fuchsin in the staining solution is 0.2 g / L to 1 g / L, and the concentration of methyl green is 0.1 g / L to 1 g / L. As mentioned above, these concentrations represent the working concentrations of the staining agents. That is, when the staining solution at these concentrations is mixed with the sample to be tested, the concentration of the dye in the staining solution may decrease due to the dilution effect of the sample to be tested.

[0014] In a specific embodiment, in the staining solution, the concentration of acid fuchsin is 0.2 g / L, 0.5 g / L, and 1 g / L; the concentration of methyl green is 0.1 g / L, 0.5 g / L, and 1 g / L.

[0015] In a specific embodiment, the staining solution contains 0.2 g / L of acid fuchsin and 0.1 g / L of methyl green;

[0016] Alternatively, the staining solution contains acid fuchsin: 0.2 g / L and methyl green: 0.5 g / L;

[0017] Alternatively, the staining solution contains acid fuchsin: 0.2 g / L and methyl green: 1 g / L;

[0018] Alternatively, the staining solution contains acid fuchsin: 0.5 g / L and methyl green: 0.1 g / L;

[0019] Alternatively, the staining solution contains acid fuchsin: 0.5 g / L and methyl green: 0.5 g / L;

[0020] Alternatively, the staining solution contains acid fuchsin: 0.5 g / L and methyl green: 1 g / L;

[0021] Alternatively, the staining solution contains acid fuchsin: 1 g / L and methyl green: 0.1 g / L;

[0022] Alternatively, the staining solution contains acid fuchsin: 1 g / L and methyl green: 0.5 g / L;

[0023] Alternatively, the staining solution contains acid fuchsin: 1 g / L and methyl green: 1 g / L.

[0024] The staining solution of the present invention also includes Triton. Triton is a surfactant that promotes the dissolution of acid fuchsin and methyl green in the staining solution and facilitates the staining of cells by the staining solution. The present invention screened the types of surfactants in the staining solution, and the results showed that Triton was more effective than other surfactants in improving the staining effect, ensuring that the nuclei of epithelial cells or white blood cells were stained while leaving live Trichomonas unstained, thereby effectively distinguishing them.

[0025] Furthermore, the present invention also optimizes the concentration of Triton in the dye, and the results show that when the volume fraction of Triton is 0.1 vol% to 1 vol%, the dyeing effect is better than that at other concentrations.

[0026] In the dye, the role of the defoamer is to change the surface tension of the bubbles and quickly eliminate the bubbles and foam generated in the system. The present invention has found that adding a defoamer can avoid the generation of bubbles during film making, making the field of vision clearer and avoiding misjudgment. In order to avoid the interference of the defoamer on the dye and the surfactant, the present invention screened the defoamers and finally selected the D248 defoamer based on the defoaming effect and the dyeing effect. In the embodiment of the present invention, the concentration of the D248 defoamer is 0.01 to 0.2 g / L; preferably, the concentration of the D248 defoamer is 0.1 g / L.

[0027] Because the pH value of urogenital samples is unstable and varies significantly between individuals, the staining solution of the present invention optimizes and adjusts its buffer to maintain stable detection results in the complex pH environment of urogenital samples. When citrate or acetate is used as a buffer, the staining effect is more stable. In the staining solution of the present invention, the buffer is a citrate buffer or an acetate buffer.

[0028] In some embodiments, the staining solution includes 1 g / L methyl green, 1 g / L acid fuchsin, 3.157 g / L sodium acetate·3H2O, 1.02 vol% glacial acetic acid, 1 vol% Triton X-ray distillate, and 0.1 g / L D248.

[0029] In other embodiments, the staining solution includes: 1 g / L methyl green, 1 g / L acid fuchsin, 27.3 g / L citric acid monohydrate, 20.6 g / L trisodium citrate dihydrate, 1 vol% Triton and 0.1 g / L D248.

[0030] In other embodiments, the staining solution includes: 0.5 g / L methyl green, 0.5 g / L acid fuchsin, 27.3 g / L citric acid monohydrate, 20.6 g / L trisodium citrate dihydrate, 1 vol% Triton and 0.1 g / L D248.

[0031] In other embodiments, the staining solution includes: 1 g / L methyl green, 1 g / L acid fuchsin, 27.3 g / L citric acid monohydrate, 20.6 g / L trisodium citrate dihydrate, 0.1 vol% Triton and 0.1 g / L D248.

[0032] In other embodiments, the staining solution includes: 0.1 g / L methyl green, 0.2 g / L acid fuchsin, 27.3 g / L citric acid monohydrate, 20.6 g / L trisodium citrate dihydrate, 0.1 vol% Triton and 0.1 g / L D248.

[0033] In other embodiments, the staining solution includes: 1 g / L methyl green, 1 g / L acid fuchsin, 27.3 g / L citric acid monohydrate, 20.6 g / L trisodium citrate dihydrate and 1 vol% Triton.

[0034] The present invention also provides a method for preparing the dyeing solution as described above,

[0035] The method comprises dissolving each component separately, then adding the components into the buffer solution, and filtering to obtain the dye solution;

[0036] Alternatively, the method comprises dissolving each component and buffer salt in water to a fixed volume, and then filtering to obtain the dyeing solution.

[0037] That is, in the preparation method described in the present invention, each component can be prepared into a mother liquor separately, and then the mother liquors can be mixed, fixed to volume, and filtered to obtain a dyeing solution. Alternatively, each component can be directly weighed and added to water to dissolve, fixed to volume, and filtered to obtain a dyeing solution.

[0038] As a feasibility case, the dissolving of each component separately includes: dissolving the methyl green to 2 times of the working concentration to the saturation concentration; dissolving the acid fuchsin to 2 times of the working concentration to the saturation concentration; dissolving the Triton to 2 times of the working concentration to the saturation concentration; dissolving the buffer salt to 2 times of the working concentration to the saturation concentration; and dissolving the defoaming agent to 2 times of the working concentration to the saturation concentration.

[0039] In the present invention, the dissolved solution is water. The saturated concentration refers to the saturated concentration at normal temperature and pressure.

[0040] The present invention also provides the use of the aforementioned staining solution in preparing a staining reagent for detecting urogenital system samples.

[0041] In the present invention, the urogenital system sample includes at least one of vaginal secretions, cervical swab, urine or urethral swab.

[0042] In the present invention, the urogenital system sample is a female urogenital system sample, and can also be a male urogenital system sample, wherein the detection targets include epithelial cells, leukocytes, Trichomonas, and / or Candida. Any sample containing the aforementioned detection targets can be stained with the staining solution of the present invention. As a feasibility case, in the embodiments of the present invention, vaginal secretion samples were used to verify the effectiveness.

[0043] Furthermore, the present invention also provides a method for detecting urogenital system samples, which comprises mixing the urogenital system sample with the aforementioned staining solution and then observing under a microscope.

[0044] In an embodiment of the present invention, the volume ratio of the urogenital system sample to the staining solution is (1-5):1. Taking vaginal secretions as an example, the volume ratio of the urogenital system sample to the staining solution is 1:1, 2:1, 3:1, 4:1, or 5:1. In the embodiment, a volume ratio of 3:1 is used as an example for verification.

[0045] In the embodiment of the present invention, the observation is performed immediately after the mixing or within 5 minutes.

[0046] This staining solution is simple and quick to use, using the wet mount staining method. It stains the sample immediately after mixing with the solution, without the need for waiting. However, experiments have shown that observation within 5 minutes will not result in a decrease in the effect.

[0047] In the embodiments of the present invention, the microscopic observation is performed using wet mount staining. The wet mount staining microscopy of the present invention comprises: mixing a sample with a stain, dropping the mixture onto a glass slide, covering the slide with a coverslip, and then placing the sample under a microscope for examination. The volume of the mixture dropped onto the slide is 40 μL, and the magnification for microscopic examination is 40×.

[0048] In the present invention, in the urogenital system sample:

[0049] The nuclei of leukocytes are stained blue-green, and the cytoplasm is colorless;

[0050] Epithelial cytoplasm stained red, and nuclei stained dark blue;

[0051] Live trichomonas are not stained, while dead trichomonas are stained red;

[0052] Candida does not stain.

[0053] Unstained sections can be easily distinguished by their morphology. For example, Candida species are small and oval, with a diameter of 3-6 microns, while live Trichomonas species are conical or pear-shaped (10-20 microns in long diameter and 5-15 microns in wide diameter).

[0054] In the present invention, after the sample is mixed with the stain, it is necessary to prepare the slide. The preparation mainly includes the steps of adding the sample dropwise onto the slide and then covering it with a coverslip. The preparation can be performed manually or by an automatic instrument for automatic mixing and preparation. The detection method of the present invention also includes the steps of taking pictures and identifying the pictures. The identification of the pictures is performed manually or by AI image recognition software. In the present invention, the pictures can be taken manually or by automatic microscopy, and the present invention does not limit this.

[0055] The testing of urogenital system samples described in the present invention may be for diagnostic or non-diagnostic purposes, and the present invention is not limited thereto. For example, when the testing is for non-diagnostic purposes, the testing may be solely for the purpose of better distinguishing various cells or pathogens, thereby better utilizing software to distinguish them. Alternatively, the testing may be performed for scientific research purposes, or for experimental purposes.

[0056] The present invention uses two different dyes—an acidic dye and a basic dye—to create a rapid staining solution. Through wet-slide staining, this solution quickly stains the nuclei of white blood cells in a sample blue-green, leaving the cytoplasm unstained; stains the nuclei of epithelial cells dark blue, with the cytoplasm unstained; leaves live Trichomonas unstained, while dead Trichomonas remain unstained; and leaves mold unstained, enabling clear and accurate differentiation of various formed elements. After staining, the structures of epithelial cells, white blood cells, Trichomonas, and Candida species are clearly defined, with distinct morphological and color differences. Combined with AI image analysis, this solution improves the accuracy of Trichomonas identification, eliminates instrument-based Trichomonas review rates, and enables fully automated interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The staining effect of the staining agent of Example 1 on the vaginal secretion sample is shown;

[0058] Figure 2 The staining effect of the staining agent of Example 2 on the vaginal secretion sample is shown;

[0059] Figure 3 The staining effect of the staining agent of Example 3 on the vaginal secretion sample is shown;

[0060] Figure 4 The staining effect of the staining agent of Example 4 on the vaginal secretion sample is shown;

[0061] Figure 5 The staining effect of the staining agent of Example 5 on the vaginal secretion sample is shown;

[0062] Figure 6 The staining effect of the staining agent of Example 6 on the vaginal secretion sample is shown;

[0063] Figure 7 The figure shows the dyeing effect of the dye in Comparative Example 1 on the vaginal secretion sample;

[0064] Figure 8 The figure shows the dyeing effect of the dye in comparative example 2 on the vaginal secretion sample;

[0065] Figure 9 The figure shows the dyeing effect of the dye of Comparative Example 3 on the vaginal secretion sample;

[0066] Figure 10 Shows the dyeing effect of different dyeing time;

[0067] Figure 11 The comparison of dyeing effect after dyeing agent placement is shown;

[0068] Figure 12 Shows the comparison of the staining effects of different stains on samples;

[0069] Figure 13 The results show the comparison of different staining agents on the staining effect of cultured Trichomonas;

[0070] Figure 14 The results show the comparison of the detection results of Trichomonas positive samples by automatic staining recognition;

[0071] Figure 15 The figure shows the comparison of the detection results of fully automatic staining recognition on samples with a large number of white blood cells;

[0072] Figure 16 Shown are photos of all 20 samples identified by the software. DETAILED DESCRIPTION

[0073] The present invention provides a staining solution and a staining method for urogenital system samples. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0074] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0075] The terms "include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0076] The term "and / or" describes the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0077] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0078] The numerical ranges and parameters used in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0079] Some cases are described in the Examples and Comparative Examples of the present invention. The Examples show some implementations of the present invention. However, this does not mean that the effects of the present invention can be achieved only in these cases. In fact, any concentration of each component between the two endpoint values shown in the Examples can achieve a good dyeing effect. The three Comparative Examples and several control samples only list some cases with poor results in the tests. In addition, many attempts have been made in the research and development process, such as screening out components that can better coordinate with acid fuchsin and methyl green from numerous surfactants, defoamers or buffers, and making the dyeing effect more obvious by adjusting the concentration or proportion of each component. However, the effects of these attempts are all inferior to those of Examples 1 to 5, and will not be described in detail here.

[0080] The test materials used in the present invention are all common commercially available products. The vaginal secretion samples are from a hospital in Henan Province that have been confirmed to be positive for Trichomonas and leukocytes. The fully automatic recognition software used is the recognition software that comes with the company's fully automatic reproductive secretion analyzer (Yu Medical Device Registration No. 20222220134).

[0081] It should be understood that in the various embodiments of the present application, the order of the sequence numbers of the above-mentioned processes does not necessarily indicate the order of execution. Some or all of the steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The present invention is further described below with reference to the following embodiments:

[0082] Example 1

[0083] Preparation of staining solution: To prepare 100 mL of staining solution, weigh 100 mg of methyl green, 100 mg of acid fuchsin, and 0.3157 g of sodium acetate·3H2O, add 1 mL of Triton X-ray dilution, 1.02 mL of glacial acetic acid, and 10 mg of D248. Add purified water to make up to 100 mL, stir and mix, and filter to obtain the vaginal secretion sample rapid staining solution.

[0084] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide for preparation. Observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained red, the nucleus is stained blue, the white blood cell nucleus is stained blue-green, Trichomonas is not stained or is stained light red, and Candida is not stained. Figure 1 shown.

[0085] Example 2

[0086] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 100 mg of methyl green, 100 mg of acid fuchsin, 2.73 g of citric acid monohydrate, and 2.06 g of trisodium citrate dihydrate, respectively. Add 1 mL of Triton X-ray distilled water, add 10 mg of D248, and dilute to 100 mL with purified water. Stir and mix thoroughly, then filter to obtain a rapid staining solution for vaginal secretions.

[0087] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide for preparation. Observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained red, the nucleus is stained blue, the white blood cell nucleus is stained blue-green, and the trichomonas is not stained or stained light red. Figure 2 shown.

[0088] Example 3

[0089] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 50 mg of methyl green, 50 mg of acid fuchsin, 2.73 g of citric acid monohydrate, and 2.06 g of trisodium citrate dihydrate, respectively, adding 1 mL of Triton X-ray distilled water, adding 10 mg of D248, and adding purified water to make up to 100 mL. Stir and mix thoroughly, and then filter to obtain a rapid staining solution for vaginal secretions.

[0090] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide for preparation. Observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained red, the nucleus is stained blue, the white blood cell nucleus is stained blue-green, Trichomonas is not stained or is stained light red, and Candida is not stained. Figure 3 shown.

[0091] Example 4

[0092] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 100 mg of methyl green, 100 mg of acid fuchsin, 2.73 g of citric acid monohydrate, and 2.06 g of trisodium citrate dihydrate, respectively, adding 0.1 mL of Triton X-ray distilled water, adding 10 mg of D248, and diluting the volume to 100 mL with purified water. Stir and mix thoroughly, and then filter to obtain a rapid staining solution for vaginal secretions.

[0093] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide, observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained red and the nucleus is stained blue, the white blood cell nucleus is stained blue-green, the small round cell nucleus is stained blue and the cytoplasm is red, and Candida is not stained. Figure 4 shown.

[0094] Example 5

[0095] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 10 mg of methyl green, 20 mg of acid fuchsin, 2.73 g of citric acid monohydrate, and 2.06 g of trisodium citrate dihydrate, respectively, adding 1 mL of Triton X-ray distilled water, adding 10 mg of D248, and adding purified water to make up to 100 mL. Stir and mix thoroughly, and then filter to obtain a rapid staining solution for vaginal secretions.

[0096] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide, observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained light pink, the nucleus is stained blue, the white blood cell nucleus is stained green or blue-green, and trichomonas is not stained. Figure 5 shown.

[0097] Example 6

[0098] Preparation of staining solution: To prepare 100 mL of staining solution, weigh 100 mg of methyl green, 100 mg of acid fuchsin, and 0.3157 g of sodium acetate·3H2O, add 1 mL of Triton X-ray distillate and 1.02 mL of glacial acetic acid, and dilute to 100 mL with purified water. Stir and mix thoroughly, then filter to obtain the vaginal secretion sample rapid staining solution.

[0099] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide for preparation. Observe under a 40x microscope, and you can clearly see that the epithelial cytoplasm is stained red, the nucleus is stained blue, the white blood cell nucleus is stained blue-green, Trichomonas is not stained or is stained light red, and Candida is not stained. Figure 6 shown.

[0100] Comparative Example 1

[0101] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 100 mg of methyl green, 100 mg of acid fuchsin, 0.3157 g of sodium acetate·3H2O, and 1.02 mL of glacial acetic acid, add 10 mg of D248, and dilute to 100 mL with purified water. Stir and mix thoroughly, then filter to obtain the vaginal secretion sample rapid staining solution.

[0102] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide for preparation. Observe under a 40x microscope and observe that the epithelial cytoplasm is stained red but the nucleus is not stained. The leukocytes and trichomonas are not stained and their morphology is difficult to distinguish. There is a large amount of blue undissolved blue dye under the microscope. Figure 7 shown.

[0103] Comparative Example 2

[0104] Preparation of staining solution: To prepare 100 mL of staining solution, weigh 100 mg of methyl green, 100 mg of acid fuchsin, and 0.3157 g of sodium acetate·3H2O, add 0.2 g of SDS, 1.02 mL of glacial acetic acid, and 10 mg of D248. Add purified water to make up to 100 mL, stir and mix, and filter to obtain the vaginal secretion sample rapid staining solution.

[0105] Sample staining method: Take a vaginal secretion sample containing a large number of white blood cells and mix it with the cultured Trichomonas in a ratio of 1:1. Take 45uL of the mixed sample, add 15ul of the above staining solution, mix well, take 40uL and drop it on the slide to prepare the slide. Observe it under a 40x microscope and you will see that only part of the epithelial cytoplasm is stained blue, while white blood cells and Trichomonas are stained blue. The cell structure is not clear and white blood cells and Trichomonas cannot be distinguished. Figure 8 shown.

[0106] Comparative Example 3

[0107] Preparation of staining solution: Prepare 100 mL of staining solution by weighing 10 mg of methyl green, 10 mg of acid fuchsin, 0.3157 g of sodium acetate.3H2O, and 1.02 mL of glacial acetic acid, respectively. Add 1 mL of Triton X-ray dilution and 10 mg of D248, and dilute to 100 mL with purified water. Stir and mix thoroughly, then filter to obtain a rapid staining solution for vaginal secretions.

[0108] Sample staining method: aspirate 45uL of vaginal secretion sample, add 15ul of the above staining solution, mix well, aspirate 40uL and drop it on a glass slide, observe under a 40x microscope, and observe that the epithelial cytoplasm is not stained, the nucleus is stained light green, and the white blood cell nucleus is stained green. Figure 9 shown.

[0109] Dyeing liquid performance test

[0110] The staining solution of Example 1 was used as the test substance. The sample staining method included: aspirating 45 μL of vaginal secretion sample, adding 15 μL of the above staining solution, mixing, aspirating 40 μL and dropping it onto a glass slide for preparation, and observing with a 40x microscope.

[0111] 1. Influence of dyeing time

[0112] like Figure 10 As shown: After stirring and mixing, immediate observation clearly shows that the epithelial cytoplasm is stained red, the nucleus is stained blue, the white blood cell nucleus is stained blue-green, Trichomonas is not stained or is stained light red, and Candida is not stained. In the same field of view, continuous observation for 5 minutes shows that the staining of each component does not change significantly with the extension of time after the sample is stained. This shows that after the staining solution is mixed with the sample, the staining results can be observed directly without waiting, and observation within at least 5 minutes will not affect the accuracy of the test.

[0113] 2. Stability of dyeing solution

[0114] After the staining solution of Example 1 was prepared, the samples were immediately stained, and staining verification was performed using a Trichomonas-negative sample, cultured Trichomonas, and a mixed sample (Trichomonas-negative sample and cultured Trichomonas were mixed at a ratio of 1:1).

[0115] After that, the staining solution was placed at room temperature for 4 months. In addition, fresh Trichomonas-negative samples, cultured Trichomonas, and mixed samples (Trichomonas-negative samples and cultured Trichomonas were mixed in a ratio of 1:1) were selected for staining verification.

[0116] like Figure 11 As shown, the freshly prepared staining solution stained a Trichomonas-negative sample. All epithelial cells in the sample stained red, with the cytoplasm stained red and the nuclei blue-green. White blood cells were all stained, with the nuclei appearing green. In a cultured Trichomonas sample, the Trichomonas were unstained, and some dead Trichomonas appeared light red. In a mixed sample, the Trichomonas appeared light red or colorless, while the white blood cell nuclei appeared green, making the Trichomonas easily distinguishable from the white blood cells. This indicates that after four months of storage at room temperature, the prepared staining solution showed no difference in staining between the samples and the fresh solution. White blood cells and epithelial cells showed 100% staining, while the Trichomonas remained unstained or stained light red, making them easily distinguishable from the white blood cells.

[0117] 3. Comparison of dyeing effects

[0118] The staining solution of Example 1 was used as the test substance, and the following staining solution was used as the control:

[0119] Safflower staining solution: Add 90 mL of purified water to a container, then add 0.1 g of Safflower O (Safflower T), 0.8345 g of potassium dihydrogen phosphate, and 0.1910 g of disodium hydrogen phosphate in sequence, stir until fully dissolved, and adjust the volume to 100 mL.

[0120] Methylene blue staining solution: Add 90 mL of purified water to a container, then add 0.1 g of methylene blue, 0.8345 g of potassium dihydrogen phosphate, and 0.1910 g of disodium hydrogen phosphate in sequence, stir until fully dissolved, and adjust the volume to 100 mL.

[0121] Compare to CN201510484688.X staining solution: mixed staining solution of safflower yellow and methylene blue.

[0122] Compare CN116625778A staining solution: a mixed staining solution of basic fuchsin and methylene blue.

[0123] Mix the staining solution with the sample in a ratio of 1:3 and prepare slides for observation under a high-power microscope.

[0124] Vaginal secretion samples positive for Trichomonas were selected and subcultured and purified using a special Trichomonas culture medium. Purely cultured Trichomonas were stained with safranin staining solution, methylene blue staining solution, and the staining solution of Example 1, respectively. Results: After staining with safranin and methylene blue, live Trichomonas were not stained, while dead Trichomonas were stained. Vaginal secretion samples containing a large number of white blood cells (Trichomonas negative) and Trichomonas positive samples were selected and stained with safranin, methylene blue, and the staining solution of Example 1, respectively. The results are as follows: Figure 12 After staining with safranin and methylene blue, a large number of white blood cells in the samples (Trichomonas negative and Trichomonas positive) were not stained, so it was impossible to distinguish Trichomonas from white blood cells through the image. After staining with the staining solution of the present invention, Trichomonas were not stained, and the white blood cell nuclei were all stained blue-green, and the various formed elements were clearly distinguished.

[0125] like Figure 13 As shown in FIG, adopt basic fuchsin+methylene blue staining solution to dye pure cultured Trichomonas, observe that Trichomonas does not dye, and dead Trichomonas dyes red.But adopt this staining solution to dye the vaginal secretion sample with a large amount of leukocytes, have some leukocytes do not dye equally.The sample positive for Trichomonas is dyed, some leukocytes in the sample do not dye, and Trichomonas does not dye either, and can not effectively distinguish whether there is Trichomonas.After adopting the staining solution of the present invention to dye the sample positive for Trichomonas, Trichomonas does not dye, and the leukocyte nucleus all dyes into bluish green, and each formed element is obviously distinguished.

[0126] 4. Fully automatic dye identification and verification

[0127] Select vaginal secretion samples with positive trichomonas and vaginal secretion samples with more leukocytes (trichomonas negative), respectively, and use safranin and Example 1 staining solution on a fully automatic instrument for preparation, staining and photography. Use AI image recognition software to identify the stained pictures. The results are as follows Figure 14 As shown: the image of the Trichomonas positive sample stained with safranin did not identify Trichomonas, while the image of the Trichomonas positive sample stained with the dyeing solution of the present invention identified Trichomonas.

[0128] In the vaginal secretion sample with more leukocytes (negative for Trichomonas), after being stained with safranin, the AI recognition software identified two Trichomonas (misidentification); in the picture stained with the dye of the present invention, the AI software did not identify Trichomonas ( Figure 15 ).

[0129] 5. Software recognition effect of 20 vaginal secretion samples

[0130] Twenty vaginal secretion samples were selected for testing and verification, including three Trichomonas-positive samples. The samples were stained with the staining solution of the present invention, photographed, and identified using fully automatic recognition software. After identification, the numbers of white blood cells, epithelial cells, and Trichomonas were manually reviewed and recorded. The staining rates of white blood cells and Trichomonas were analyzed and compared. The software recognition results were compared with those of manual detection, and the detection rate (the number of cells identified by the software divided by the number of cells identified manually) was calculated.

[0131] The results are as follows Figure 16 The 20 samples showed a 100% leukocyte staining rate, no trichomonad staining, and a 100% epithelial cell staining rate. Compared with manual analysis, the software's identification of each component showed a 100% detection rate for trichomonads, a 100% detection rate for Candida species, a >90% leukocyte detection rate, and a >90% epithelial cell detection rate.

[0132] Table 1

[0133]

[0134]

[0135] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A staining solution for genital secretion samples, characterized in that: The invention comprises acid fuchsin and methyl green, wherein the mass ratio of acid fuchsin to methyl green is (20-100):(10-100).

2. The dyeing solution according to claim 1, wherein The mass ratio of the acid fuchsin to methyl green is (1-2):

1.

3. The dyeing solution according to claim 1 or 2, characterized in that The invention also comprises Triton, D248 defoaming agent and buffer; the buffer is citrate buffer or acetate buffer.

4. The dyeing solution according to any one of claims 1 to 3, characterized in that in: The concentration of acid fuchsin is 0.2g / L to 1g / L; The concentration of methyl green is 0.1g / L to 1g / L; The volume fraction of Triton is 0.1 vol% to 1 vol%; The concentration of D248 defoamer is 0.01~0.2g / L.

5. The method for preparing the dyeing solution according to any one of claims 1 to 4, The method comprises dissolving each component separately, then adding the components into the buffer solution, and filtering to obtain the dye solution; Alternatively, the method comprises dissolving each component and buffer salt in water to a fixed volume, and then filtering to obtain the dyeing solution.

6. Use of the staining solution according to any one of claims 1 to 4 in preparing a staining reagent for detecting urogenital system samples.

7. The use according to claim 6, characterized in that The urogenital system sample includes at least one of vaginal secretions, cervical swab, urine or urethral swab.

8. The use according to claim 6, characterized in that The detection targets include at least one of epithelial cells, leukocytes, Trichomonas and / or Candida.

9. A method for detecting a urogenital system sample, comprising mixing the urogenital system sample with the staining solution according to any one of claims 1 to 4, preparing a slide and observing the slide under a microscope; Alternatively, the method comprises mixing a urogenital system sample with the staining solution according to any one of claims 1 to 4, photographing the sample after preparation, and then using AI image recognition software to identify the stained image.

10. The detection method according to claim 9, characterized in that: The volume ratio of the urogenital system sample to the staining solution according to any one of claims 1 to 4 is (1 to 5):1.

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