A giemsa staining solution and its application

Abstract

This invention relates to the field of medical testing, and particularly to a Wright-Gymsza staining solution and its application. The Wright-Gymsza staining solution of this invention comprises staining solution A and staining solution B. Staining solution A mainly consists of eosin Y, azurite-eosin A, methylene blue, cell fixative, and stabilizer, while staining solution B is a buffer solution. Experiments have shown that the Wright-Gymsza staining solution of this invention can stain normally for 27 months after preparation, solving the technical problems of uneven staining and dye residue on the staining platform after a period of time after opening.

Description

Technical Field

[0001] This invention relates to the field of medical testing, and in particular to a Wright-Gymsa staining solution used in blood slide staining machines and its application. Background Technology

[0002] Wright-Gymsa staining solution is a composite staining agent composed of Wright's staining solution and Giemsa staining solution.

[0003] Wright's stain was originally developed to better observe the morphology of blood cells and is widely used in hematology due to its ability to clearly display various cellular structures. Giemsa stain is very effective for staining cell nuclei and parasites. Later, it was discovered that mixing Wright's and Giemsa stains could combine the advantages of both, leading to the development of Wright-Gemsa stain, which plays an important role in clinical testing and research.

[0004] In hematological testing, Wright-Gymsa staining is one of the most fundamental staining methods. From early disease diagnosis, such as malaria (which allows observation of the morphology of the malaria parasite within red blood cells), to modern diagnosis and research of complex hematological diseases, Wright-Gymsa staining has played an irreplaceable role.

[0005] In the field of pathology, for cytological examinations, such as the examination of specimens like sputum, urine, and biopsy cells, Wright-Giemsa staining can help identify tumor cells and inflammatory cells, providing important clues for preliminary diagnosis and further investigation. For example, in sputum cytology, Wright-Giemsa staining can be used to screen for lung cancer and observe whether the sample contains cancer cells.

[0006] The Wright-Gymsar staining machine is an automated device designed based on the Wright-Gymsar staining principle. It uses a mechanical device to evenly cover the staining solution on the slide and completes the staining process according to the set time, temperature and rinsing procedures to achieve standardized and efficient staining results.

[0007] Compared to manual dyeing, the dyeing machine can strictly follow the set parameters for dyeing, reducing the dyeing differences caused by different operators and different operating habits.

[0008] However, the Wright-Gymsa staining solution used in slide staining machines has the following limitations:

[0009] 1) Uneven staining may occur after the staining solution has been used for a certain period of time since it was opened;

[0010] 2) When using online, if dyeing is not performed for a long time, dye residue will dry on the dyeing platform, causing the first piece of background to be dirty in the next dyeing. Summary of the Invention

[0011] In view of this, the present invention provides a Wright-Gymsa staining solution for use in a blood slide staining machine and its application, which solves the technical problems of uneven staining and dye residue on the staining platform after a period of time after opening the bottle.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] This invention provides a combination of Wright-Gymsa staining solutions, comprising staining solution A and staining solution B;

[0014] The staining solution A includes: eosin Y, azurite eosin A, methylene blue, surfactant, stabilizer, and protectant;

[0015] The staining solution B includes: disodium hydrogen phosphate, potassium dihydrogen phosphate, and a preservative;

[0016] The stabilizer includes at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90;

[0017] The protective agent includes glycerin.

[0018] In some specific embodiments of the present invention, in the staining solution A of the above-described Wright-Gymsa staining solution combination:

[0019] The content of eosin Y is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L;

[0020] The content of the azurite A is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L;

[0021] The methylene blue content is 2 g / L to 8 g / L, and can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L or 8 g / L;

[0022] The surfactant content is 0.5 g / L to 2 g / L, and can be 0.5 g / L, 1 g / L, 1.5 g / L or 2 g / L;

[0023] The content of the stabilizer is 0.5 g / L to 2 g / L, and can be 0.5 g / L, 1 g / L, 1.5 g / L or 2 g / L;

[0024] The content of the protective agent is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L.

[0025] In some specific embodiments of the present invention, in the staining solution B of the above-described Wright-Gymsa staining solution combination:

[0026] The content of disodium hydrogen phosphate is 5 g / L to 15 g / L, and can be 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L or 15 g / L;

[0027] The content of potassium dihydrogen phosphate is 3 g / L to 9 g / L, and can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or 9 g / L;

[0028] The content of the preservative is 0.5 g / L to 1 g / L, and can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L.

[0029] In some specific embodiments of the present invention, the solvent of staining solution A in the above-mentioned Wright-Gymsa staining solution combination is methanol.

[0030] In some specific embodiments of the present invention, the solvent of the staining solution B in the above-mentioned Wright-Gymsa staining solution combination is purified water.

[0031] This invention also provides a method for preparing a combination of Wright-Gymsa staining solutions, comprising:

[0032] Eosin Y, Azurite A, Methylene Blue, surfactant, stabilizer, protectant and solvent are mixed to obtain staining solution A;

[0033] Next, mix disodium hydrogen phosphate, potassium dihydrogen phosphate, preservative and solvent to obtain staining solution B, which is the Wright-Gymsa staining solution combination;

[0034] The stabilizer includes at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90;

[0035] The protective agent includes glycerin.

[0036] In some specific embodiments of the present invention, the staining solution A prepared by the above method includes:

[0037] The content of eosin Y is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L;

[0038] The content of the azurite A is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L;

[0039] The methylene blue content is 2 g / L to 8 g / L, and can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L or 8 g / L;

[0040] The surfactant content is 0.5 g / L to 2 g / L, and can be 0.5 g / L, 1 g / L, 1.5 g / L or 2 g / L;

[0041] The content of the stabilizer is 0.5 g / L to 2 g / L, and can be 0.5 g / L, 1 g / L, 1.5 g / L or 2 g / L;

[0042] The content of the protective agent is 1 g / L to 5 g / L, and can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L.

[0043] In some specific embodiments of the present invention, the staining solution B described in the above preparation method:

[0044] The content of disodium hydrogen phosphate is 5 g / L to 15 g / L, and can be 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L or 15 g / L;

[0045] The content of potassium dihydrogen phosphate is 3 g / L to 9 g / L, and can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or 9 g / L;

[0046] The content of the preservative is 0.5 g / L to 1 g / L, and can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L.

[0047] In some specific embodiments of the present invention, the solvent of the dye A prepared by the above method is methanol.

[0048] In some specific embodiments of the present invention, the solvent of the staining agent B in the above preparation method is purified water.

[0049] The present invention also provides a combination of Wright-Gymsa staining solutions prepared by the above preparation method.

[0050] The present invention also provides a kit comprising acceptable excipients or adjuvants and:

[0051] (i) The above combination of Wright-Gymsa staining solutions; or

[0052] (ii) The combination of Wright-Gymsa staining solutions prepared by the above method.

[0053] In some specific embodiments of the present invention, the kit described above is a sperm cell staining kit, a blood cell staining kit, a gynecological leukorrhea smear staining kit, or a bone marrow smear staining kit.

[0054] The present invention also provides an apparatus comprising an acceptable component and:

[0055] (i) The above combination of Wright-Gymsa staining solutions; or

[0056] (ii) The combination of Wright-Gymsa staining solutions prepared by the above method.

[0057] In some specific embodiments of the present invention, the above-mentioned device is a cell smear staining instrument.

[0058] The present invention also provides a staining method based on any of the following staining methods:

[0059] (i) The above combination of Wright-Gymsa staining solutions; or

[0060] (ii) The combination of Wright-Gymsa staining solutions prepared by the above method; or

[0061] (iii) The above-mentioned reagent kit; or

[0062] (iv) The aforementioned device.

[0063] The present invention has the following effects:

[0064] Experiments have shown that the Wright-Gymsza staining solution of the present invention can stain normally for 27 months after preparation, without uneven staining or dye residue on the staining platform. Furthermore, leukocyte nuclei on smears stained with the Wright-Gymsza staining solution of the present invention show better and easier staining. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0066] Figure 1 Showing microscopic images taken on March 5, 2019;

[0067] Figure 2 Showing microscopic images taken on April 8, 2019;

[0068] Figure 3 Showing microscopic images taken on May 7, 2019;

[0069] Figure 4 Showing microscopic images taken on June 4, 2019;

[0070] Figure 5Showing microscopic images taken on September 5, 2019;

[0071] Figure 6 Showing microscopic images taken on December 5, 2019;

[0072] Figure 7 Showing microscopic images taken on March 6, 2020;

[0073] Figure 8 Showing microscopic images taken on June 4, 2020;

[0074] Figure 9 Showing microscopic images taken on September 7, 2020;

[0075] Figure 10 Showing microscopic images taken on December 7, 2020;

[0076] Figure 11 Showing microscopic images taken on March 5, 2021;

[0077] Figure 12 This is a microscopic examination photo taken on June 4, 2021. Detailed Implementation

[0078] This invention discloses a Wright-Gymsa staining solution for use in blood slide staining machines and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0079] This invention provides a Wright-Gymsa staining solution formulation, mainly used for staining blood smears, and is used in conjunction with the staining process of the 26 series blood cell morphology analyzer. It can also be used for staining bone marrow smears, vaginal discharge samples, exfoliated cells, etc.

[0080] The Wright-Gymsard staining solution of this invention includes staining solution A and staining solution B. Staining solution A mainly consists of eosin Y, azurite-eosin A, methylene blue, cell fixative, and stabilizer, while staining solution B is a buffer solution. During instrument staining, the staining steps are completed sequentially via a conveyor belt, and after staining, the slides can be dried, stored in a cassette, and pushed to the slide reader.

[0081] Wright-Gymsard staining primarily consists of eosin Y, azurite A, and methylene blue. These dyes bind to different components in the cell, producing specific color reactions. Eosinophilic granules stain pink, basophilic granules stain purplish-red, and neutral substances stain pale blue.

[0082] Specifically, the formula for the Wright-Gymsa staining solution of the present invention is as follows:

[0083] Dyeing solution A:

[0084] Eosin Y: 1 g / L~5 g / L;

[0085] Azure-Erythrine A: 1 g / L~5 g / L;

[0086] Methylene blue: 2 g / L~8 g / L;

[0087] Surfactant: 0.5 g / L~2 g / L;

[0088] Stabilizer: 0.5 g / L~2 g / L;

[0089] Protectant: 1 g / L~5 g / L;

[0090] Solvent: Methanol.

[0091] Staining solution B:

[0092] Disodium hydrogen phosphate: 5 g / L~15 g / L;

[0093] Potassium dihydrogen phosphate: 3 g / L~9 g / L;

[0094] Preservatives: 0.5 g / L~1 g / L;

[0095] Solvent: Purified water.

[0096] The surfactant may be Triton, the stabilizer may be polyvinylpyrrolidone K30 and / or polyvinylpyrrolidone K90, the protectant may be glycerin, and the preservative may be ProClin300.

[0097] One method for preparing the Wright-Gymsa staining reagent of the present invention includes:

[0098] Eosin Y, Azurite A, Methylene Blue, surfactant, stabilizer and protectant are mixed to obtain staining solution A; then disodium hydrogen phosphate, potassium dihydrogen phosphate and preservative are mixed to obtain staining solution B, which is the Wright-Gymsa staining solution.

[0099] One specific method of using the Wright-Gymsa staining reagent of the present invention is as follows: Following the instrument's instruction manual, connect Wright-Gymsa solution A and Wright-Gymsa solution B to the corresponding interfaces of the instrument; the instrument will automatically aspirate and stain. In some specific embodiments, manual operation is also possible; the specific steps are as follows:

[0100] 1) Add an appropriate amount of Wright-Gymsa A solution to cover the smear and stain at room temperature for 1 minute.

[0101] 2) Add Wright-Gymsa B solution to solution A (the amount added is 1.5 to 3 times that of solution A), mix thoroughly, and stain at room temperature for 3 to 10 minutes (depending on the nature of the smear; the staining time for blood smears can be shorter, and for bone marrow smears it varies depending on the amount of cells).

[0102] The following are reference rules for identifying various analytes in smears or sections stained with the Wright-Gymsa staining reagent of the present invention:

[0103] 1) Staining of blood cell smears and bone marrow smears: Under a microscope, red blood cells appear pink, white blood cell cytoplasm shows the unique colors of various cells, cell nuclei are stained purplish-red, and chromatin is clear;

[0104] 2) Vaginal discharge smear staining: ① White blood cells: The staining effect is the same as that of blood smears. Generally, the number of white blood cells indicates the degree of inflammation; ② Trichomonas: Trichomonas are mostly pear-shaped, round, oval or irregular in shape. The plasma cells are stained sky blue. The nucleus is small, date-shaped, and often located on one side; ③ Fungi: Stained dark blue, spores or pseudohyphae can be seen; ④ Clue cells: Stained blue;

[0105] 3) Staining of exfoliated cell smears: The cell nucleus is stained purplish-red, the nucleolus is stained blue, and the cytoplasm varies in basophilia depending on the degree of differentiation, showing different shades of blue.

[0106] To better implement this invention, the following precautions should be taken regarding the smears:

[0107] 1) Blood smear: Fresh whole blood or EDTA anticoagulated blood is required. The blood smear prepared by the instrument will be automatically dried and put into the staining mode. Blood smears prepared by hand should be shaken in the air to dry as quickly as possible.

[0108] 2) Bone marrow smear: Smear well-collected bone marrow immediately and dry it quickly after smearing to prevent cell deformation;

[0109] 3) Vaginal discharge smear: After taking a fresh specimen, make a smear. The smear should be fixed with flame or alcohol as soon as possible to prevent cell deformation.

[0110] 4) Exfoliated cell smear: After taking the specimen, smear it and fix it. Different specimens require different fixation methods, including natural drying and wet slide fixation.

[0111] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0112] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0113] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0114] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0115] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0116] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0117] The azure eosin A involved in this invention is called Azure A eosinate, and its CAS number is 62298-43-5.

[0118] The present invention will be further illustrated below with reference to the embodiments.

[0119] Example

[0120] 1. Reagent preparation

[0121] Dyeing solution A:

[0122] Eosin Y: 2.5 g / L;

[0123] Azure-Erythrine A: 3 g / L;

[0124] Methylene blue: 6 g / L;

[0125] Triathon: 1 g / L;

[0126] K30 and K90 together are 1 g / L, where K30 is 0.7 g / L and K90 is 0.3 g / L;

[0127] Glycerin: 3 g / L;

[0128] Solvent: Methanol.

[0129] Staining solution B:

[0130] Disodium hydrogen phosphate: 7.1 g / L;

[0131] Potassium dihydrogen phosphate: 6.8 g / L;

[0132] ProClin300: 0.8 g / L;

[0133] Solvent: Purified water.

[0134] 2. Operating Procedures

[0135] Operate the instrument according to the instruction manual, connecting Wright-Gymsa A solution and Wright-Gymsa B solution to the corresponding interfaces on the instrument. The instrument will automatically aspirate and stain the samples. Rinse off the staining solution with running water and examine under a microscope after drying.

[0136] Example of effect

[0137] To confirm whether the Wright-Gymsa staining solution has a shelf life of 24 months, an experiment was conducted between March 4, 2019 and May 6, 2021.

[0138] 1. Test Content

[0139] 1) Prepare 12 sets (20 mL / set) of Reggie staining solution according to the method in the example, and store them separately;

[0140] 2) Take one set of staining solution at each of the following months: 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, and 27 months. Use fresh anticoagulated blood to push the slides onto the staining machine for staining.

[0141] 3) Take images on the film reader;

[0142] 4) Record photos for each month.

[0143] 2. Test Records

[0144] The microscopic images from March 5, 2019, April 8, 2019, May 7, 2019, June 4, 2029, September 5, 2029, December 5, 2019, March 6, 2020, June 4, 2020, September 7, 2020, December 7, 2020, March 5, 2021, and June 4, 2021 are shown in the following order. Figures 1-12 As shown.

[0145] 3. Experimental Conclusions

[0146] The staining solution can stain normally for 27 months.

[0147] 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 combination of Wright-Gymsa staining solutions, characterized in that, Includes staining solution A and staining solution B; The staining solution A includes: eosin Y, azurite eosin A, methylene blue, surfactant, stabilizer, and protectant; The staining solution B includes: disodium hydrogen phosphate, potassium dihydrogen phosphate, and a preservative; The stabilizer includes at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90; The protective agent includes glycerin.

2. The Wright-Gymsa staining solution combination as described in claim 1, characterized in that: In the staining solution A, the content of eosin Y is 1 g / L to 5 g / L, the content of azurite eosin A is 1 g / L to 5 g / L, the content of methylene blue is 2 g / L to 8 g / L, the content of surfactant is 0.5 g / L to 2 g / L, the content of stabilizer is 0.5 g / L to 2 g / L, and the content of protectant is 1 g / L to 5 g / L. In the staining solution B, the content of disodium hydrogen phosphate is 5 g / L to 15 g / L, the content of potassium dihydrogen phosphate is 3 g / L to 9 g / L, and the content of preservative is 0.5 g / L to 1 g / L.

3. A method for preparing the Wright-Gymsa staining solution combination, characterized in that, include: Eosin Y, Azurite A, Methylene Blue, surfactant, stabilizer, protectant and solvent are mixed to obtain staining solution A; Next, mix disodium hydrogen phosphate, potassium dihydrogen phosphate, preservative and solvent to obtain staining solution B, which is the Wright-Gymsa staining solution combination; The stabilizer includes at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90; The protective agent includes glycerin.

4. The preparation method according to claim 3, characterized in that: In the staining solution A, the content of eosin Y is 1 g / L to 5 g / L, the content of azurite eosin A is 1 g / L to 5 g / L, the content of methylene blue is 2 g / L to 8 g / L, the content of surfactant is 0.5 g / L to 2 g / L, the content of stabilizer is 0.5 g / L to 2 g / L, and the content of protectant is 1 g / L to 5 g / L. In the staining solution B, the content of disodium hydrogen phosphate is 5 g / L to 15 g / L, the content of potassium dihydrogen phosphate is 3 g / L to 9 g / L, and the content of preservative is 0.5 g / L to 1 g / L.

5. The Wright-Gymsa staining solution combination prepared by the preparation method as described in claim 3 or 4.

6. A reagent kit, characterized in that, Including acceptable excipients or adjuvants and: (i) The Wright-Gymsa staining solution combination as described in claim 1 or 2; or (ii) The Wright-Gymsa staining solution combination prepared by the preparation method as described in claim 3 or 4.

7. The kit according to claim 6, characterized in that, The kit is a sperm cell staining kit, a blood cell staining kit, a gynecological leukorrhea smear staining kit, or a bone marrow smear staining kit.

8. An apparatus, characterized in that, Including acceptable components and: (i) The Wright-Gymsa staining solution combination as described in claim 1 or 2; or (ii) The Wright-Gymsa staining solution combination prepared by the preparation method as described in claim 3 or 4.

9. The apparatus as claimed in claim 8, characterized in that, The device is a cell smear staining instrument.

10. A staining method not for disease diagnosis or treatment purposes, characterized in that, Based on any of the following staining methods: (i) The Wright-Gymsa staining solution combination as described in claim 1 or 2; or (ii) The Wright-Gymsa staining solution combination prepared by the method described in claim 3 or 4; or (iii) The kit as described in claim 6 or 7; or (iv) The apparatus as described in claim 8 or 9.

Images