Single-step staining reagent preparation and visible component detection method for mammal blood detection

Abstract

The invention relates to a method for preparing a single-step staining reagent for mammal blood detection and detecting visible components. The single-step staining reagent comprises an acid-base regulator, osmotic pressure regulating salt, an accelerating agent, a dye, a protective agent and water, the osmotic pressure regulating salt is used for regulating the osmotic pressure environment in the cell staining process, and regulating the osmotic pressure of the microscopic examination sample to 150-230 mosmol / kg; the pH value of the cell staining process is regulated by the pH regulator, and the pH value of the microscopic examination sample is regulated to 7.0-8.0; the accelerating agent is used for promoting the affinity of the dye and nucleic acid substances in cells; the dye is neomethylene blue, a dyeing substance is provided in the dyeing process, and the content of the neomethylene blue in a microscopic examination sample is 4.0 * 10 (-3) g / L to 5.0 * 10 (-1) g / L; the protective agent can enable protein in a cell membrane to be chemically crosslinked in the dyeing process, a three-dimensional network skeleton is formed to protect the cell membrane from being broken, and a dyeing substance can pass through the three-dimensional network skeleton.

Description

Technical Field

[0001] The present application relates to the technical field of staining for bright-field identification of mammalian blood cells, and in particular to a staining reagent and method for single-step rapid staining of mammalian blood in a cell suspension state, which can rapidly and simultaneously stain multiple cells in mammalian blood and expand the temperature range of adaptability so that different types of cells in mammalian blood can be identified and counted in more demanding environments. Background Art

[0002] Different animal species are at different stages of evolution, and the types, morphology, and number of blood cells in their peripheral blood vary. The subtypes of blood cells in mammalian blood are more diverse than those in non-mammalian blood.

[0003] The applicant filed a series of Chinese patents, such as

[0004] 1. CN2020112669290, “Cell analysis method and system and quantitative method and system”;

[0005] 2. CN2020112669182, “Cell Suspension Sample Imaging Method, System, and Kit”;

[0006] 3. CN2022104799126, “Fast focusing method for microscopic image acquisition device and microscopic image acquisition method”;

[0007] 4. CN2023116834572, “Blood white blood cell identification AI training method and computing processing and storage device”.

[0008] Using a brand-new technical solution, image processing technology based on artificial intelligence algorithms can analyze and learn images to achieve recognition and understanding of image content; and use artificial intelligence algorithms to measure the content of various formed elements in the blood, including the analysis, recognition and counting of red blood cells and white blood cells at different differentiation stages in the blood.

[0009] In recognition based on brightfield microscopic magnified images, staining is necessary to highlight the characteristics of different cell types to facilitate machine learning and recognition. Because there are many target cell types that require staining, and each cell has distinct characteristics, significant variations in staining results can lead to increased learning workload and potentially anomalies or errors in recognition.

[0010] like Figure 11In the prior art, mammalian blood staining in this application scenario is typically performed using a two-step process: staining in the first step, followed by dye curing. Sufficient time intervals are required between these two steps to maintain a good staining effect. While this two-step process provides guaranteed staining results, it also reduces sample preparation efficiency.

[0011] Improving staining efficiency while ensuring effective staining remains a technical challenge. Furthermore, in these applications, to image both red and white blood cells simultaneously after tiling, it is necessary to ensure that the red blood cells are flat within the microscopic field of view. Different species have varying red and white blood cell ratios, so achieving the appropriate dilution ratio during staining is also a technical challenge.

[0012] The applicants have also proposed a method for quantitatively measuring hemoglobin in red blood cells (RBCs) through colorimetric microscopy. This method places strict demands on image background light intensity and dye concentration. During the dilution staining process, different species and test items require different dilution factors. Different dilution factors, even varying ambient temperatures, can lead to inconsistent staining results, creating uncertainty in quantitative measurements.

[0013] Glossary:

[0014] Nucleic acid refers to the substances with genetic characteristics present in cells, namely DNA or RNA or their fragments.

[0015] Osmotic pressure: Water permeates through a semipermeable membrane, with enzyme-containing water on one side and a solution on the other. The pressure applied to the solution side to prevent water from moving is called osmotic pressure. The osmotic pressure stops water movement because the pressure is equal to the chemical potential energy of the water passing through the membrane. Osmotic pressure is measured in osmolarity, which is usually expressed in milliosmoles of solute per kilogram of solvent.

[0016] The CAS numbers of some substances in this application are as follows:

[0017] Substance name CAS Number Potassium dihydrogen phosphate 7778-77-0 New Methylene Blue 229-516-6 water 7732-18-5

[0018] (CAS Registry Number or CASNumber, CASRn, CAS#), also known as CAS accession number or CAS registration number, is a unique digital identification number for a substance (compound, polymer material, biological sequence, mixture or alloy). Summary of the Invention

[0019] In the present application, the inventors proposed a single-step staining reagent for mammalian blood testing, which can complete the staining of various cells in one step and can also simultaneously achieve mammalian blood staining and dilution.

[0020] The technical solution of the present application is a single-step staining reagent for mammalian blood testing, which is used to prepare microscopic samples for blood microscopic testing. The microscopic sample is prepared by adding the above-mentioned single-step staining reagent for mammalian blood testing to a blood sample; the above-mentioned single-step staining reagent for mammalian blood testing includes an acid-base regulator, an osmotic pressure regulating salt, a dye accelerator, a dye, a protective agent and water; the osmotic pressure regulating salt regulates the osmotic pressure environment of the cell staining process to adjust the osmotic pressure of the microscopic sample to "150mosmol / kg to 230mosmol / kg"; the acid-base regulator The agent adjusts the pH value of the cell staining process and adjusts the pH value of the microscopic sample to 7.4 to 8.0; the dye accelerator promotes the affinity of the dye with the nucleic acid substance in the cell during the staining process; the dye provides a dyeing substance during the staining process, and the dye is new methylene blue. The content of new methylene blue in the microscopic sample is 4.0×10^(-3)g / L~5.0×10^(-1)g / L; the protective agent can chemically cross-link the protein inside the cell membrane during the staining process, forming a three-dimensional network skeleton to protect the cell membrane from rupture, and the dye can pass through the three-dimensional network skeleton.

[0021] The above-mentioned osmotic pressure regulating salt includes sodium chloride, and the content of sodium chloride in the above-mentioned microscopic examination sample is 2.2×10^(-2)mol / L to 3.5×10^(-2)mol / L.

[0022] The above-mentioned acid-base regulator includes any one or more of disodium hydrogen phosphate dodecahydrate or potassium dihydrogen phosphate; the content of the acid-base regulator in the above-mentioned microscopic examination sample is 2.0×10^(-2)mol / L~1.0×10^(-1)mol / L.

[0023] The dyeing accelerator includes dipotassium ethylenediaminetetraacetate, and the content of dipotassium ethylenediaminetetraacetate in the microscopic sample is 2.0×10^(-4)mol / L to 2.0×10^(-3)mol / L.

[0024] The protective agent includes one or more of formaldehyde, paraformaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, glutaraldehyde, and phenol.

[0025] The content of the protective agent in the microscopic sample is 1.4x10^(-3)mol / L to 5.3x10^(-2)mol / L.

[0026] The above-mentioned single-step staining reagent for mammalian blood testing further includes a preservative 950, and the volume proportion of the preservative 950 is 0.1%.

[0027] A single-step staining reagent for mammalian blood testing, which is dried to separate the water to form a dry powder staining reagent.

[0028] The technical solution of the present application can also be a method for preparing a single-step staining reagent for mammalian blood testing, wherein the single-step staining reagent for mammalian blood testing is used to prepare a blood test microscopic sample; water is mixed with each component to prepare a single-step staining reagent for mammalian blood testing with a volume of V2; the single-step staining reagent for mammalian blood testing is used to dilute the blood sample N times, the volume of the microscopic sample is V3, and the content of component A in the microscopic sample is K3; the volume of the single-step staining reagent for mammalian blood testing is V2, and the content of component A is K2; the content of component A K2 == K3×N / (N-1). The single-step staining reagent for mammalian blood testing is used to dilute the blood sample N times, where N is greater than 50 and N is less than 800.

[0029] The preparation method of the above-mentioned single-step staining reagent for mammalian blood detection includes any of the following features: TA1: Component A of the single-step staining reagent for mammalian blood detection is an osmotic pressure regulating salt, the osmotic pressure regulating salt is sodium chloride, and the content is (2.2×10^(-2)mol / L to 3.5×10^(-2)mol / L)×N / (N-1); TA2: Component A of the single-step staining reagent for mammalian blood detection is a dyeing accelerator, and the dyeing accelerator includes dipotassium ethylenediaminetetraacetate, and the content is (2.0×10^(-4)mol / L to 2.0×10^(-3)mol / L)×N / (N-1); TA3: Component A of the single-step staining reagent for mammalian blood testing is a dye, with a content of (4.0×10^(-3)g / L~5.0×10^(-1)g / L)×N / (N-1); TA4: Component A of the single-step staining reagent for mammalian blood testing is a protective agent, with a content of (1.4x10^(-3)mol / L~5.3x10^(-2)mol / L)×N / (N-1); TA5: Component A of the single-step staining reagent for mammalian blood testing is an acid-base regulator, with a content of 2.0×10^(-2)mol / L~1.0×10^(-1)mol / L×N / (N-1).

[0030] The technical solution of the present application may also be a method for detecting formed elements in mammalian blood, wherein the method comprises photographing a microscopic sample to obtain an image, the microscopic sample being prepared using the single-step staining reagent for mammalian blood detection, and the microscopic sample image being photographed between time T1 and time T2 after the microscopic sample is prepared. T1 is 0.5 to 5 minutes, and T2 is 10 to 30 minutes.

[0031] The technical solution of the present application can also be a mammalian blood formed element detection device, which photographs a microscopic sample to obtain an image, and the microscopic sample is prepared using the above-mentioned mammalian blood detection single-step staining reagent.

[0032] The technical solution of the present application may also be a method for AI training for detecting formed elements in mammalian blood. During the AI ​​training process, images are obtained by photographing microscopic samples, wherein the microscopic samples are prepared using the single-step staining reagent for detecting mammalian blood according to any one of claims 1 to 8. During the preparation of the microscopic samples, the mixing temperature is between 12°C and 47°C. The images of the microscopic samples are captured between time T1 and time T2 after the microscopic sample preparation is completed; T1 is 0.5 to 5 minutes; and T2 is 10 to 30 minutes.

[0033] One of the technical effects of the above technical solution is: through the clever design of the environment and formula combination, the single dye component in the formula can quickly cross the cell membrane and enter the cell with the help of the ion concentration difference inside and outside the cell, so that the dye can easily enter the cell membrane and combine with the nuclear material in the cell, completing the rapid staining of the cell nuclear material; a single reagent addition can quickly complete the staining of multiple different types of cells, realizing single-step staining of a single dye staining reagent.

[0034] One of the technical effects of the above technical solution is that the osmotic pressure regulating salt can provide appropriate osmotic pressure and provide a suitable atmosphere for rapid single-step dyeing.

[0035] One of the technical effects of the above technical solution is that the acid-base regulator can provide appropriate pH and provide a suitable atmosphere for rapid single-step dyeing.

[0036] One of the technical effects of the above technical solution is that the dyeing accelerator promotes the affinity between the dye and the nucleic acid substance in the cell during the dyeing process.

[0037] One of the technical effects of the above technical solution is that two dyeing accelerators, sodium chloride and dipotassium ethylenediaminetetraacetic acid, are designed to promote dyeing at the same time, so that single-step dyeing can be achieved.

[0038] One of the technical effects of the above technical solution is that the aldehyde-containing substances or phenol in the protective agent will react with the protein on the surface of the cell membrane, causing the protein to denature and inactivate, and the dye will more easily penetrate the cell membrane, achieving single-step rapid staining.

[0039] One of the technical effects of the above technical solution is that the protective agent can chemically cross-link the proteins inside the cell membrane during the dyeing process, forming a three-dimensional network skeleton to protect the cell membrane from rupture, and the dye can pass through the three-dimensional network skeleton.

[0040] One of the technical effects of the above technical solution is that the concentration of the protective agent is set within an appropriate range, and it is a one-step staining reagent that is very suitable for mammalian blood.

[0041] One of the technical effects of the above technical solution is that the preservative 950 can increase the shelf life of the reagent.

[0042] One of the technical effects of the above technical solution is that the dry powder staining reagent is easy to carry and transport.

[0043] One of the technical effects of the above technical solution is that in the single-step staining reagent preparation method for mammalian blood testing, the staining reagent volume can be flexibly set according to the volume requirements of the microscopic examination sample, and the staining reagent volume can be prepared reversely according to the needs, which is convenient for operation. More importantly, the concentration of the staining reagent components can be kept at an appropriate level at different staining reagent volumes, thereby ensuring the consistency of the staining effect.

[0044] One of the technical effects of the above technical solution is that in order to achieve the same staining effect, the components in the liquid dye at different dilution ratios are different. Especially in the hemoglobin quantitative test process, the dye has an impact on the measurement, so it is necessary to accurately maintain the content of various dye components in the unit volume of microscopic examination sample consistent.

[0045] One of the technical effects of the above technical solution is that: while dyeing all target shaped components in a single step, dilution can also be achieved at the same time, and the dyeing and dilution of multiple shaped components can be completed in one step, further improving the dyeing efficiency.

[0046] One of the technical benefits of the above technical solution is that the temperature range for preparing and photographing microscopic samples is 12 degrees Celsius to 47 degrees Celsius, which is greater than the temperature tolerance of conventional reagents at room temperature of 25 degrees Celsius, and can adapt to a wider range of ambient temperatures. It can also adapt to staining at lower temperatures.

[0047] One of the technical effects of the above technical solution is that the image of the microscopic sample is captured between time T1 and time T2 after the microscopic sample is prepared; T1 is 0.5 minutes to 5 minutes, and T2 is 10 minutes to 30 minutes, which can maintain the staining effect for a longer time, leaving sufficient time for photography.

[0048] One of the technical benefits of the above technical solution is that the AI ​​training method for detecting formed elements in mammalian blood uses the above-mentioned single-step staining reagent for mammalian blood testing to prepare microscopic examination samples, which is highly efficient and can complete the staining and dilution of multiple formed elements in one step. It also ensures the consistency of staining across all AI training samples, thereby improving the accuracy of AI recognition. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1This is a schematic diagram of a method for detecting formed elements in mammalian blood;

[0050] Figure 2 This is a schematic diagram of the AI ​​training method for detecting formed elements in mammalian blood;

[0051] Figure 3 It is a schematic diagram of the classification of single-step staining reagent formulas for mammalian blood testing;

[0052] Figure 4 This is one of several schematic diagrams of components of a single-step staining reagent for mammalian blood testing;

[0053] Figure 5 This is a collection of images of some blood cells obtained by microscopically magnifying the stained samples after staining of canine blood cells using Example 1, Example 2, and Example 3;

[0054] Figure 6 This is a collection of pictures of some blood cells observed after comparative example 1 and comparative example 2 were applied to canine blood cells for staining;

[0055] Figure 7 A comparison of the image set of blood cells stained in Example 2 and the image set of blood cells stained in the two-step method in the prior art;

[0056] Figure 8 This is one of the schematic diagrams of the method for preparing a single-step staining reagent for mammalian blood testing;

[0057] Figure 9 This is the second schematic diagram of the method for preparing a single-step staining reagent for mammalian blood testing;

[0058] Figure 10 This is the third schematic diagram of the method for preparing a single-step staining reagent for mammalian blood testing;

[0059] Figure 11 This is a schematic diagram of the mammalian blood staining process in the prior art;

[0060] Figure 12 This is the second schematic table of components of multiple embodiments of a single-step staining reagent for mammalian blood testing;

[0061] Figure 13 This is a table showing the components of several comparative examples of single-step staining reagents for mammalian blood testing. DETAILED DESCRIPTION

[0062] The following is a further detailed description of the present application in conjunction with the accompanying drawings. It should be noted that the following is a description of a preferred embodiment of the present invention and does not constitute any limitation to the present invention. The description of the preferred embodiment of the present invention is only for the purpose of illustrating the general principles of the present invention. The numbers such as "first", "second", "A", and "B" involved in the present invention are only for the convenience of explanation and do not represent a temporal or spatial order.

[0063] like Figure 1 and Figure 2 Whether it is the detection of formed elements in mammalian blood or the AI ​​training method for detecting formed elements in mammalian blood, dyeing is the basic step, and dyeing is the basis for detection and AI training. The effect and efficiency of dyeing directly determine the operating efficiency and effect of the subsequent system. If the dyeing consistency is not good, the accuracy of AI recognition will also be reduced. As a chemical process, dyeing substances are extremely sensitive to the environment. They are not only affected by their own components, but also easily affected by environmental conditions, such as temperature. When the temperature conditions change, how to ensure dyeing consistency is a technical problem to be solved. In order to solidify the dyeing effect, the existing technology usually adopts the following methods: Figure 11 Or similar two-step staining, the efficiency is low.

[0064] like Figure 3 As shown, a formula of a single-step staining reagent for mammalian blood testing is used to prepare a microscopic sample for blood microscopic testing. The microscopic sample is formed by adding the above-mentioned single-step staining reagent for mammalian blood testing to a blood sample; the above-mentioned single-step staining reagent for mammalian blood testing includes an osmotic pressure regulating salt, an acid-base regulating agent, a dye accelerator, a dye, a protective agent and water; the osmotic pressure regulating salt regulates the osmotic pressure environment of the cell staining process, adjusting the osmotic pressure of the microscopic sample to "150mosmol / kg-230mosmol / kg"; the acid-base regulating agent regulates the pH value of the cell staining process, adjusting the pH value of the microscopic sample to 7.4 to 8.0.

[0065] During the staining process, the dye accelerator promotes the affinity of the dye for intracellular nucleic acids. The dye provides a dyeing substance during the staining process. The dye is new methylene blue, and the content of new methylene blue in microscopic samples is 4.0×10^(-3)g / ml to 5.0×10^(-1)g / mL. During the staining process, the protective agent chemically cross-links proteins within the cell membrane, forming a three-dimensional network that protects the cell wall from rupture and allows the dye to pass through the three-dimensional network. The above-mentioned osmotic pressure regulating salt includes sodium chloride. The above-mentioned acid-base adjusting agents include disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate.

[0066] The above-mentioned osmotic pressure regulating salt includes sodium chloride, and the content of sodium chloride in the above-mentioned microscopic examination sample is 2.2×10^(-2)mol / L~3.5×10^(-2)mol / L; the above-mentioned dyeing accelerator includes dipotassium ethylenediaminetetraacetate, and the content of dipotassium ethylenediaminetetraacetate in the above-mentioned microscopic examination sample is 2.0×10^(-4)mol / L~2.0×10^(-3)mol / L.

[0067] The above-mentioned acid-base regulator includes any one or more of disodium hydrogen phosphate dodecahydrate or potassium dihydrogen phosphate; the content of the acid-base regulator in the above-mentioned microscopic examination sample is 2.0×10^(-2)mol / L~1.0×10^(-1)mol / L.

[0068] The protective agent includes one or more of formaldehyde, paraformaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, glutaraldehyde, and phenol. The protective agent is present in the microscopic sample at a concentration of 1.4 x 10^(-3) mol / L to 5.3 x 10^(-2) mol / L. The single-step staining reagent for mammalian blood testing also includes preservative 950, with the volume percentage of preservative 950 being 0.1%.

[0069] like Figure 4 , lists 3 example dyeing conditions and key parameters, such as Figure 12 , lists the dyeing conditions and key parameters of Example 4 to Example 16; Figure 13 , the dyeing conditions and key parameters of 3 comparative examples are listed. Figure 4 、 Figure 12 、 Figure 13 In the table, only the key components and environmental conditions are listed. Figure 4 and Figure 12 It can be seen that all 15 embodiments can achieve normal staining of leukocytes and facilitate leukocyte classification.

[0070] Figure 5 The dyeing pictures of Examples 1 to 3 are shown in the figure. Example 1 is carried out at 12 degrees Celsius. Although the temperature is less than 0.5 times the general temperature of 25 degrees Celsius, the lower the temperature, the lower the diffusion activity of the dye. However, in this application, due to the mutual coordination of the various components, the dyeing at 12 degrees Celsius can still reach a level sufficient for the machine to identify the dyeing consistency.

[0071] like Figure 6 , in the dyeing effect of comparative example 1 and comparative example 2, compared with comparative example 1 and comparative example 2, the temperature is 28 degrees Celsius, which is consistent with the temperature condition of embodiment 3, but the dyeing effect of comparative example 1 and comparative example 2 is as Figure 6 As shown. Figure 5Comparing the dyeing effects of Example 1 and Example 3, it is obvious that the dyeing of Example 1 and Example 2 is insufficient and cannot support subsequent recognition and calculation.

[0072] Further, if Figure 7 As shown, Example 2 in the present application and the two-step staining method in the prior art were selected for comparison. It can be seen that the staining effects are equivalent, which shows that the one-step staining reagent and method in the present application can completely replace the two-step staining method.

[0073] like Figure 8 and Figure 9 , a method for preparing a single-step staining reagent for mammalian blood testing, wherein the single-step staining reagent for mammalian blood testing is used to prepare a blood sample for microscopic examination; water is mixed with each component to prepare a single-step staining reagent for mammalian blood testing with a volume of V2; the single-step staining reagent for mammalian blood testing is used to dilute the blood sample by N times, the volume of the microscopic examination sample is V3, and the content of component A in the microscopic examination sample is K3; the volume of the single-step staining reagent for mammalian blood testing is V2, and the content of component A is K2; the content of component A K2 = K3×N / (N-1).

[0074] The calculation process is as follows: the volume of the microscopic specimen is V3, the volume of the single-step staining reagent for mammalian blood testing is V2, and the blood specimen volume is V3 - V2; the dilution factor N = V3 / (V3-V2); V3 = V2 × N / (N-1). V3 × K3 = V2 × K2; K2 = V3 × K3 / V2 = K3 × V3 / V2 = K3 × N / (N-1). This allows for precise calculation of reagent volumes based on the required microscopic specimen volume. Differences can be ignored at infinite dilution factors, and configuration errors can be precisely controlled at smaller dilution factors.

[0075] The above-mentioned single-step staining reagent for mammalian blood testing is used to dilute the blood sample N times, where N is greater than 50 and N is less than 800. The preparation method of the single-step staining reagent for mammalian blood testing is as follows: Component A of the above-mentioned single-step staining reagent for mammalian blood testing is an osmotic pressure regulating salt, which is sodium chloride, and its content is (2.2×10^(-2)mol / L~3.5×10^(-2)mol / L)×N / (N-1); Component A of the above-mentioned single-step staining reagent for mammalian blood testing is a dyeing accelerator, which includes dipotassium ethylenediaminetetraacetate, and its content is (2.0×10^(-4)mol / L~2.0×10^(-3)mol / L)×N / (N-1); Component A of the single-step staining reagent for detecting blood of mammals is a dye, and its content is (4.0×10^(-3)g / L~5.0×10^(-1)g / L)×N / (N-1); component A of the single-step staining reagent for detecting blood of mammals is a protective agent, and its content is (1.4x10^(-3)mol / L~5.3x10^(-2)mol / L)×N / (N-1); component A of the single-step staining reagent for detecting blood of mammals is an acid-base regulator, and its content is 2.0×10^(-2)mol / L~1.0×10^(-1)mol / L×N / (N-1).

[0076] like Figure 10 The mammalian blood detection single-step staining reagent is dried to separate water to form a dry powder staining reagent. When used, water and the dry powder staining reagent are mixed to form the mammalian blood detection single-step staining reagent, which is mixed with blood to form a microscopic examination sample.

[0077] like Figure 1 As shown, a method for detecting formed elements in mammalian blood is provided. The method comprises photographing a microscopic sample to obtain an image. The microscopic sample is prepared using the single-step staining reagent described above for mammalian blood testing. The image of the microscopic sample is taken between time T1 and time T2 after the microscopic sample is prepared. T1 is 0.5 to 5 minutes, and T2 is 10 to 30 minutes. The temperature range for preparing and photographing the microscopic sample is 18 to 47 degrees Celsius.

[0078] In an embodiment of a mammalian blood formed element detection device, the mammalian blood formed element detection device photographs a microscopic sample to obtain an image, and the microscopic sample is prepared using the mammalian blood detection single-step staining reagent.

[0079] like Figure 2As shown, a method for AI training for detecting formed elements in mammalian blood is provided. During the AI ​​training process, images are obtained by photographing microscopic samples, which are prepared using the single-step staining reagent for detecting mammalian blood according to any one of claims 1 to 8. During the preparation of the microscopic samples, the mixing temperature is between 18°C ​​and 47°C. The images of the microscopic samples are captured between time T1 and time T2 after the microscopic sample preparation; T1 is 0.5 to 5 minutes, and T2 is 10 to 30 minutes.

[0080] Although the present invention is illustrated and described based on the preferred embodiment and several alternatives, the invention is not limited by the specific description in this specification. Other additional replacement or equivalent components can also be used to practice the present invention.

Claims

1. A single-step staining reagent for mammalian blood testing, used for preparing blood microscopic examination samples, characterized in that: The microscopic examination sample is formed by adding the mammalian blood detection single-step staining reagent to a blood sample; The single-step staining reagent for mammalian blood testing comprises an acid-base regulator, an osmotic pressure regulating salt, a dye accelerator, a dye, a protective agent and water; Osmotic pressure regulating salt adjusts the osmotic pressure environment during cell staining and adjusts the osmotic pressure of microscopic samples to "150mosmol / kg~230mosmol / kg"; Acid-base regulators adjust the pH value of the cell staining process and adjust the pH value of the microscopic specimen to 7.4 to 8.0; Dye accelerators promote the affinity of dyes to nucleic acid substances in cells during the dyeing process; The dye provides dyeing substances during the staining process. The dye is new methylene blue. The content of new methylene blue in the microscopic specimen is 4.0×10^(-3)g / L~5.0×10^(-1)g / L. During the staining process, the protective agent can chemically cross-link the proteins inside the cell membrane, forming a three-dimensional network skeleton to protect the cell membrane from rupture, and the dye can pass through the three-dimensional network skeleton.

2. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: The osmotic pressure regulating salt includes sodium chloride, and the content of sodium chloride in the microscopic examination sample is 2.2×10^(-2)mol / L to 3.5×10^(-2)mol / L.

3. The single-step staining reagent for mammalian blood detection according to claim 1 or 2, characterized in that: The acid-base regulator includes any one or more of disodium hydrogen phosphate dodecahydrate or potassium dihydrogen phosphate; the content of the acid-base regulator in the microscopic sample is 2.0×10^(-2)mol / L to 1.0×10^(-1)mol / L.

4. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: The dyeing accelerator includes dipotassium ethylenediaminetetraacetate, and the content of dipotassium ethylenediaminetetraacetate in the microscopic sample is 2.0×10^(-4)mol / L to 2.0×10^(-3)mol / L.

5. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: The protective agent includes one or more of formaldehyde, paraformaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, glutaraldehyde, and phenol.

6. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: The content of the protective agent in the microscopic sample is 1.4x10^(-3)mol / L to 5.3x10^(-2)mol / L.

7. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: The invention also includes a preservative 950, and the volume proportion of the preservative 950 is 0.1%.

8. The single-step staining reagent for mammalian blood detection according to claim 1, characterized in that: A single-step staining reagent for mammalian blood testing, which is dried to separate the water to form a dry powder staining reagent.

9. A method for preparing a single-step staining reagent for mammalian blood testing, characterized in that: The mammalian blood detection single-step staining reagent is used to prepare blood detection microscopic examination samples; Water was mixed with each component to prepare a single-step staining reagent for mammalian blood detection with a volume of V2; The mammalian blood detection single-step staining reagent is used to dilute the blood sample N times, The volume of the microscopic sample is V3, and the content of component A in the microscopic sample is K3; The volume of the single-step staining reagent for mammalian blood detection is V2, and the content of component A is K2; The content of component A K2 = K3 × N / (N-1).

10. The method for preparing a single-step staining reagent for mammalian blood testing according to claim 9, characterized in that: N is greater than 50 and N is less than 800.

11. The method for preparing a single-step staining reagent for mammalian blood testing according to claim 10, characterized in that: Include any of the following characteristics; TA1: Component A of the mammalian blood test single-step staining reagent is an osmotic pressure regulating salt, which is sodium chloride, with a content of (2.2×10^(-2)mol / L to 3.5×10^(-2)mol / L)×N / (N-1); TA2: Component A of the mammalian blood test single-step staining reagent is a dye accelerator, which includes dipotassium ethylenediaminetetraacetate in an amount of (2.0×10^(-4)mol / L to 2.0×10^(-3)mol / L)×N / (N-1); TA3: Component A of the mammalian blood detection single-step staining reagent is a dye, with a content of (4.0×10^(-3)g / L to 5.0×10^(-1)g / L)×N / (N-1); TA4: Component A of the mammalian blood detection single-step staining reagent is a protective agent, with a content of (1.4x10^(-3)mol / L to 5.3x10^(-2)mol / L)×N / (N-1); TA5: Component A of the mammalian blood detection single-step staining reagent is an acid-base regulator with a content of 2.0×10^(-2)mol / L to 1.0×10^(-1)mol / L×N / (N-1).

12. A method for detecting formed elements in mammalian blood, characterized in that: The method for detecting formed elements in mammalian blood comprises photographing a microscopic sample to obtain an image, wherein the microscopic sample is prepared using the single-step staining reagent for detecting mammalian blood according to any one of claims 1 to 8, and the microscopic sample image is photographed between time T1 and time T2 after the microscopic sample is prepared.

13. The method for detecting formed elements in mammalian blood according to claim 12, wherein: The T1 is 0.5 minutes to 5 minutes; the T2 is 10 minutes to 30 minutes.

14. The method for detecting formed elements in mammalian blood according to claim 12, wherein: The temperature range for preparing and photographing the microscopic samples is 12 degrees Celsius to 47 degrees Celsius.

15. A device for detecting formed components in mammalian blood, characterized in that: The mammalian blood formed element detection device photographs a microscopic sample to obtain an image, and the microscopic sample is prepared using the mammalian blood detection single-step staining reagent according to any one of claims 1 to 8.

16. A method for AI training for detecting formed elements in mammalian blood, characterized in that: During the AI ​​training process, microscopic samples are photographed to obtain images, and the microscopic samples are prepared using the single-step staining reagent for mammalian blood detection according to any one of claims 1 to 8.

17. The AI ​​training method for detecting formed elements in mammalian blood according to claim 16, characterized in that: During the preparation of the microscopic examination samples, the mixing temperature condition is 12 degrees Celsius to 47 degrees Celsius.

18. The AI ​​training method for detecting formed elements in mammalian blood according to claim 16, characterized in that: The microscopic sample image is taken between time T1 and time T2 after the microscopic sample is prepared; The T1 is 0.5 minutes to 5 minutes; the T2 is 10 minutes to 30 minutes.

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