CN118758823A - A kit for white blood cell classification and counting and its application - Google Patents

Abstract

The present invention discloses a kit for leukocyte classification and counting and its application, wherein the dye is allowed to enter the cell through a permeabilizing agent, and the cell nucleus is stained by adjusting the dye ratio, the pH of the dye solution and the concentration of the fixing agent, while the cytoplasm also presents different colors, and the five classifications of leukocytes are realized by combining the characteristics of cell size, lobed morphology of the cell nucleus and the color of the cytoplasm. The present invention is a kit and a staining method that are simple to prepare, easy to use, stable, high in staining efficiency, obvious in classification characteristics, and can realize the five classifications of leukocytes at the same time, and is suitable for automatic identification and classification and counting of formed elements in samples by cell morphology analysis technology based on the principle of machine vision.

Description

A kit for white blood cell classification and counting and its application

Technical Field

The invention belongs to the technical field of a kit for cell classification and counting, and relates to a kit for white blood cell classification and counting and an application thereof.

Background Art

Classification and clinical significance of white blood cells: White blood cells are a type of colorless, spherical, nucleated blood cells, which can be divided into two categories according to their morphology: granulocytes and agranulocytes. Granular white blood cells (granulocytes) contain special dye granules, and three types of granulocytes can be distinguished by staining with Wright's dye, namely neutrophils, eosinophils and basophils; agranulocytes have no cytoplasmic granules, including monocytes and lymphocytes.

When a disease occurs in the body, the number of white blood cells often changes, so it is one of the methods for disease examination and diagnosis. (1) The most common cause of neutrophilia is infection, especially bacterial infection, and the degree of infection is often proportional to the increase in the number of white blood cells. A decrease in this type of cells is common in viral infection, long-term exposure to radiation, chemotherapy and radiotherapy of tumors, hypersplenism, autoimmune diseases, etc.; (2) Eosinophilia is seen in allergic reactions, parasitic diseases, etc., and its decrease is seen in typhoid fever, paratyphoid fever, and after the use of adrenocortical hormones; (3) Basophilia is seen in chronic myeloid leukemia, Hodgkin's disease, cancer metastasis, lead and bismuth poisoning, etc.; (4) Lymphocytosis is seen in whooping cough, infectious mononucleosis, chronic lymphocytic leukemia, measles, mumps, tuberculosis, infectious hepatitis; its decrease is often seen in the acute stage of infection, radiation sickness, cellular immune deficiency, etc.; (5) Monocytes: Increased in active tuberculosis, typhoid fever, malaria, kala-azar, recovery period of acute infectious diseases, monocytic leukemia, subacute infective endocarditis, etc.

Existing WBC counting methods:

Manual method: Using blood smear and Wright-Giemsa staining, combined with manual microscopic examination, is the "gold standard" for five-category white blood cell counting. Wright-Giemsa stain is a composite dye composed of acid dye eosin and basic dye methylene blue, which respectively combine with eosinophilic and basophilic substances in white blood cells to make them different colors, thereby distinguishing the five types of white blood cells.

Electrical impedance method: Existing fully automatic instruments can first process red blood cells with special reagents, and then use flow technology or electrical impedance counting principles to obtain the fluorescence and scattered light information or pulse signals emitted by different organelles of white blood cells to classify white blood cells into different types. The instrument method is easy to operate, fast to detect, with good repeatability, easy to standardize, and suitable for batch specimen testing.

Image method: A single-person blood cell analyzer based on POCT (point-of-care diagnosis) stains blood cells by mixing blood with a staining reagent, and then adds the stained blood cell suspension into a cavity 100-200μm high. After settling to the same level, it is photographed, and then the classification and counting are automatically completed through the image algorithm. It combines the advantages of manual and instrumental methods, does not require time-consuming and labor-intensive pictures and manual counting, and has the characteristics of no liquid path, maintenance-free, no pollution-free liquid, small size, low cost, high degree of automation, fast detection, and good repeatability.

Existing technical problems:

Traditional manual staining reagents have poor staining effects on blood cell suspensions, which can easily lead to cell rupture and make it impossible to achieve cell classification. The prior art discloses a variety of special reagents and staining solutions for blood cell analyzers, such as Patent Publication No. CN1126836A, which classifies white blood cells into 5 categories using electrical impedance method and a separate basophil channel, and Patent Publication No. CN101349644A, which classifies white blood cells into 5 categories using a fluorescent dye in combination with a blood cell analyzer. These special reagents and staining solutions cannot be used directly for staining blood cell suspensions. Patent Publication No. CN116046502A discloses a method for simultaneously staining reticulocytes and white blood cells with new methylene blue and identifying and counting them through AI. Although this method directly stains blood cell suspensions and does not The red blood cells are destroyed, but a single dye is used, the cell nucleus and part of the cell cytoplasm are blue, which is not easy to distinguish, and it is mainly used for pets; Patent Publication No. CN103398935A discloses a method for re-staining blood cells after mixing methylene blue and eosin Y. The staining principle is the same as the traditional Wright-Giemsa principle, but no smear is used. Instead, the red blood cells are destroyed by non-ionic surfactant and holes are punched in the white blood cells to promote the entry of the dye into the white blood cells. The method is identified and counted by AI. The method is simple to operate, but the cell density is low and the detection time is long. The two dyes are difficult to prepare due to different pH values and are prone to precipitation. In terms of staining effect, the amount of dye used is large, and the neutrophil and eosinophil granules are purple-red, which is not enough to distinguish; Patent Publication No. CN 116106541A discloses a method for staining a blood cell suspension, but the specific formula is unknown, and a separate basophil counting pool is required for counting; Patent Publication No. CN105074422A uses a mixture of three dyes, eosin, neo-substantially blue and crystal violet, for staining. The preparation is relatively cumbersome and the reaction temperature is between 37°C and 60°C, which requires a high temperature control effect of the instrument, and the staining effect and discrimination are not described. None of the above methods provide a description of leukocyte aggregation, which can be seen in disease causes, such as infection, malignant tumors, autoimmune diseases, infectious mononucleosis, etc. Some people believe that these factors weaken the repulsion between leukocytes or destroy the cell membrane of leukocytes, leading to leukocyte aggregation.

For the imaging method, in order to improve the efficiency of image acquisition, a larger leukocyte density is often required to reduce the image acquisition time, but the increased density will cause leukocyte aggregation, which interferes with the detection of pathogenic aggregation. The existing technology has not yet been optimized for leukocyte aggregation.

It is reported that there are at least two mechanisms that cause leukocyte aggregation: one is the presence of EDTA-dependent autoantibodies in the patient's serum, that is, the aggregation-promoting effect of EDTA anticoagulants; the other is the presence of cold antibodies in the patient's serum, that is, cold antibodies cause aggregation reactions. During leukocyte classification analysis, leukocyte aggregation will seriously affect the accuracy of counting and classification, so a staining solution that can evenly disperse leukocytes and prevent aggregation is needed.

Therefore, there is an urgent need for a special reagent for white blood cell classification and counting that is easy to use, has good stability, high staining efficiency, obvious classification characteristics and can meet the requirements of five classifications, which can be used for clinical detection of white blood cells based on machine vision principles.

Summary of the invention

Aiming at the problem that the prior art lacks a special reagent for classification and counting of leukocytes that is easy to use, has good stability, high staining efficiency, obvious classification characteristics, and can realize five-category classification based on the principle of machine vision, the present invention provides a kit for classification and counting of leukocytes and its application. By adjusting the dye ratio, pH and fixative concentration, the cell nucleus and cytoplasm are stained, and the five-category classification of leukocytes is realized by combining the characteristics of cell size, cell nuclear lobed morphology and cytoplasm color. Five types of leukocyte classification and counting can be performed simultaneously in one staining, and the cell density is high and the counting time is short. The classification and counting of 2000 leukocytes in a single sample can be completed in 2 minutes.

The purpose of the present invention is achieved through the following technical solutions:

A kit for leukocyte classification and counting, comprising a reagent B and a reagent R, wherein the components of the reagent B and the reagent R both contain a dye, a buffer, a permeabilizing agent, an osmotic pressure regulator, an anticoagulant, a preservative, and a fixative;

The specific functions of each ingredient are as follows:

The dye quickly enters the white blood cells in the liquid state, where the acidic dye combines with the eosinophilic substances (eosinophilic granules, etc.) in the white blood cells, and the basic dye combines with the basophilic substances (nucleic acids, basophilic granules, etc.), resulting in certain differences in the coloring of different white blood cells;

The buffer provides a suitable pH environment for staining;

Permeabilizing agents can bind to the phospholipid bilayer or protein on the membrane to make the phospholipid layer liquid crystal or protein conformation;

Osmotic pressure regulators provide a hypotonic environment. Under the hypotonic environment, cells swell and create gaps in the cell membrane and nuclear membrane, increasing permeability.

Anticoagulants can prevent leukocyte aggregation, stabilize the nonpolar surface of proteins, and reduce the movement of protein molecules;

Preservatives allow reagents to be preserved for a long time;

The fixative cross-links the cell surface proteins, maintaining the intact morphology of the white blood cells while fixing the stained substances inside the cells.

Specific:

A kit for leukocyte classification and counting, comprising a reagent B and a reagent R, wherein the components of the reagent B and the reagent R both contain a dye, a buffer, a permeabilizing agent, an osmotic pressure regulator, an anticoagulant, a preservative, and a fixative;

1) The composition and concentration of reagent B are:

Dye, selected from one of new methylene blue, methylene blue, brilliant tar blue, crystal violet, trypan blue, toluidine blue, and alcian blue cationic dyes, with a concentration of 0.1-0.5 g/L;

Buffer, selected from one of HEPES, Tris, MES, phosphate buffer, and citrate buffer, with a concentration of 0.01-0.10 M and a pH of 6.0-8.0;

The permeabilizing agent is selected from one of the non-ionic surfactants such as NP40, saponin, Tween 20, Tween 40, Brij35, Brij52, Brij58, Brij72, TritonX-100, TritonX-114, TritonX-165 and TritonX-305, with a concentration of 1-6 g/L;

Osmotic pressure regulator, selected from one of Na ₂ SO ₄ , NaCl, and KCl, with a concentration of 0-8 g/L;

An anti-aggregating agent selected from mannitol, with a concentration of 0.3-0.7% (v/v);

A fixative, selected from one of formaldehyde, paraformaldehyde or glutaraldehyde, with a concentration of 0.1-0.5 ml/L;

A preservative selected from one of sorbic acid, potassium sorbate, methyl paraben, propyl paraben, sodium azide, ProClin300 or BND-10, with a concentration of 0.1-0.5 mL/L;

2) The composition and concentration of reagent R are:

Dye, selected from one of eosin Y and neutral red anionic dye, with a concentration of 0.1-0.5 g/L;

Buffer, selected from one of HEPES, Tris-HCl, MES, phosphate buffer, and citrate buffer, with a concentration of 0.01-0.10 M and a pH of 6.0-8.0;

The permeabilizing agent is selected from one of the non-ionic surfactants such as NP40, saponin, Tween 20, Tween 40, Brij35, Brij52, Brij58, Brij72, TritonX-100, TritonX-114, TritonX-165 and TritonX-305, with a concentration of 1-6 g/L;

Osmotic pressure regulator, selected from one of Na ₂ SO ₄ , NaCl, and KCl, with a concentration of 0-8 g/L;

An anti-aggregating agent selected from mannitol, with a concentration of 0.3-0.7% (v/v);

A fixative, selected from one of formaldehyde, paraformaldehyde or glutaraldehyde, with a concentration of 0.1-0.5 ml/L;

A preservative selected from one of sorbic acid, potassium sorbate, methyl paraben, propyl paraben, sodium azide, ProClin300 or BND-10, with a concentration of 0.1-0.5 mL/L;

3) Preparation method: weigh the dye, buffer, permeabilizing agent, osmotic pressure regulator, anti-agglutinating agent, fixative and preservative in sequence and place them in a beaker, add ultrapure water to the set concentration, stir until completely dissolved, and filter to obtain the kit for leukocyte classification and counting.

Furthermore, in reagent B:

The dye is selected from New Methylene Blue;

The buffer is selected from a phosphate buffer consisting of Na ₂ HPO ₄ and KH ₂ PO ₄ ;

The permeabilizing agent was selected from TritonX-100;

The anti-aggregating agent is selected from mannitol;

The osmotic pressure regulator is selected from NaCl;

The fixative is selected from glutaraldehyde;

The preservative was selected from ProClin300.

Furthermore, in reagent Y:

The dye is selected from Eosin Y;

The buffer is selected from a phosphate buffer consisting of Na ₂ HPO ₄ and KH ₂ PO ₄ ;

The permeabilizing agent was selected from TritonX-100;

The anti-aggregating agent is selected from mannitol;

The osmotic pressure regulator is selected from NaCl;

The fixative is selected from glutaraldehyde;

The preservative was selected from ProClin300.

The present invention also relates to the use of the above-mentioned kit for leukocyte differential counting, which is a method for leukocyte differential counting, comprising the following steps:

1) Mixing reagent B and reagent R in a mixing ratio of (2-4):1 to obtain a mixed dye solution;

2) adding the blood sample to be tested to the mixed dye solution obtained in 1), the ratio of the mixed dye solution to the blood sample to be tested is (10-20):1, reacting and coloring immediately at room temperature, shaking and immediately adding to a counting plate, and settling for 2 minutes;

3) The counting plate after sedimentation is sent to an analyzer based on machine vision principle and AI recognition technology for automatic identification and classification counting to obtain the five-category white blood cell counting results;

The specific morphological characteristics of white blood cells after staining are as follows:

① Neutrophil cytoplasm is colorless to pale yellow, and the nucleus is bluish purple, rod-shaped or lobed;

② The eosinophil cytoplasm is stained orange to red, and the nucleus is blue-purple;

③ The cytoplasm of basophils is purple-red, and the nucleus is blue-purple;

④ The nuclei and cytoplasm of lymphocytes and monocytes are stained blue, and the lymphocytes are small. Large lymphocytes are larger than lymphocytes, but the cytoplasm accounts for a small proportion and the nuclear staining is uniform; the nucleus of monocytes is horseshoe-shaped or rod-shaped.

Furthermore, the mixing ratio of reagent B and reagent R described in step 1) is 3:1; when mixed, the amount of dye is small and precipitation will not occur in a short time, but the reagents need to be stored separately for a long time, otherwise precipitation will occur. Therefore, the present invention is set as two reagents B and R, which need to be temporarily mixed during detection.

Furthermore, the ratio of the mixed dye solution in step 2) to the blood sample to be tested is 10:1.

Compared with the prior art, the present invention has the following advantages:

1. The kit for leukocyte classification and counting of the present invention comprises two dye solutions, wherein the two dye solutions have the same components except for the different dyes (one is a cationic dye and the other is an anionic dye), and the preparation is simple; the dye concentration is low, the amount used is small, and the cost is saved; the concentration of new methylene blue and eosin Y is low, the ratio is appropriate, the dye is not easy to precipitate after mixing, and the aggregation of leukocytes can be prevented to cause result errors.

2. A kit for leukocyte classification and counting according to the present invention solves the problem of leukocyte aggregation by adding organic alcohols (mannitol). In principle, mannitol can stabilize the nonpolar surface of proteins on the leukocyte membrane, increase viscosity, and reduce molecular motion, thereby avoiding leukocyte aggregation. In addition, the addition of mannitol can also improve the uniformity of dyeing. The present invention is a kit that is simple to prepare, easy to use, has good stability, high dyeing efficiency, obvious classification characteristics, and can simultaneously achieve five classifications of leukocytes, and can be applied to leukocyte morphological analysis and inspection technology based on machine vision principles. The present invention is easy to operate, fast to color, and the cytoplasm color is clearly distinguished, and the classification is more accurate; one staining can simultaneously perform 5 types of leukocyte classification and counting, and the cell density is high, the counting time is short, and a single sample classification and counting of 2000 leukocytes only takes 2 minutes.

3. The application of the kit for leukocyte classification and counting described in the present invention allows the dye to enter the cell through a permeabilizing agent, and by adjusting the dye ratio, the pH of the dye solution and the concentration of the fixative, the reaction temperature is required to be low, so that the cell nucleus is stained, and the cytoplasm also presents different colors. Combined with the characteristics of cell size, nuclear lobation morphology and cytoplasm color, the five classifications of leukocytes are realized.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

FIG1 is a schematic diagram of normal cell staining in Example 1 of the present invention;

FIG2 is a schematic diagram of abnormal cell staining caused by low pH in Comparative Example 1 of the present invention;

FIG3 is a schematic diagram of abnormal cell staining caused by a high proportion of R dye solution in Comparative Example 2 of the present invention;

FIG4 is a schematic diagram of abnormal cell staining due to the absence of mannitol in Comparative Example 3 of the present invention;

FIG5 is a schematic diagram of abnormal cell staining due to the absence of mannitol in Comparative Example 3 of the present invention;

6 is a schematic diagram of abnormal cell staining caused by the anticoagulant selected from EDTA-2Na in Comparative Example 4 of the present invention;

FIG. 7 is a schematic diagram of dyeing in Comparative Example 5 of the present invention.

DETAILED DESCRIPTION

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all repeated three times.

Embodiment 1:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 2g/L NaCl, 0.03M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 6.0), 4g/L TritonX-100, 0.5g/L new methylene blue, 0.1mL/L glutaraldehyde; 0.5% mannitol;

Reagent R composition and concentration: 2 g/L NaCl, 0.03 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 6.0), 4 g/L Triton X-100, 0.5 g/L Eosin Y, 0.1 mL/L glutaraldehyde; 0.5% mannitol;

The mixing ratio of reagent B and R is 2:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature, and the cells are observed after sedimentation for 2 minutes.

Embodiment 2:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 0.1M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.1g/L new methylene blue, 0.3mL/L glutaraldehyde; 0.5% mannitol;

Reagent R composition and concentration: 0.1M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.1g/L Eosin Y, 0.3mL/L glutaraldehyde; 0.5% mannitol;

The mixing ratio of reagent B and R is 3:1, and the ratio of the mixed solution to blood is 15:1; after the sample is added to the counting plate, it reacts at room temperature, and the cells are observed after sedimentation for 2 minutes.

Embodiment 3:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L Triton X-100, 0.3 g/L new methylene blue, 0.5 mL/L glutaraldehyde; 0.5% mannitol;

Reagent R composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L Triton X-100, 0.3 g/L Eosin Y, 0.5 mL/L glutaraldehyde; 0.5% mannitol;

The mixing ratio of reagent B and R is 4:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature and the cells are observed after sedimentation for 2 minutes.

Embodiment 4:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 2g/L NaCl, 0.03M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.1g/L new methylene blue, 0.2mL/L glutaraldehyde; 0.3% mannitol;

Reagent R composition and concentration: 2 g/L NaCl, 0.03 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2 g/L Triton X-100, 0.1 g/L Eosin Y, 0.2 mL/L glutaraldehyde; 0.3% mannitol;

The mixing ratio of reagent B and R is 3:1, and the ratio of the mixed solution to blood is 20:1; after the sample is added to the counting plate, it reacts at room temperature, and the cells are observed after sedimentation for 2 minutes.

Embodiment 5:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 2g/L NaCl, 0.03M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.2g/L new methylene blue, 0.3mL/L glutaraldehyde; 0.7% mannitol;

Reagent R composition and concentration: 2 g/L NaCl, 0.03 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2 g/L Triton X-100, 0.2 g/L Eosin Y, 0.3 mL/L glutaraldehyde; 0.7% mannitol;

The mixing ratio of reagent B and R is 3:1, and the ratio of the mixed solution to blood is 15:1; after the sample is added to the counting plate, it reacts at room temperature, and the cells are observed after sedimentation for 2 minutes.

Embodiment 6:

A kit for white blood cell differential counting, comprising:

Reagent B composition and concentration: 1 g/L NaCl, 0.05 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 3 g/L TritonX-100, 0.2 g/L new methylene blue, 0.3 mL/L glutaraldehyde; 0.4% mannitol;

Reagent R composition and concentration: 1 g/L NaCl, 0.05 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 3 g/L Triton X-100, 0.2 g/L Eosin Y, 0.3 mL/L glutaraldehyde; 0.4% mannitol;

The mixing ratio of reagent B and R is 4:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature and the cells are observed after sedimentation for 2 minutes.

Comparative Example 1: (The difference between Comparative Example 1 and Example 1 is that the pH is different)

Reagent B composition and concentration: 2g/L NaCl, 0.03M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 5.0), 4g/L TritonX-100, 0.5g/L new methylene blue, 0.1mL/L glutaraldehyde; 0.5% mannitol;

Reagent R composition and concentration: 2 g/L NaCl, 0.03 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 5.0), 4 g/L Triton X-100, 0.5 g/L Eosin Y, 0.1 mL/L glutaraldehyde; 0.5% mannitol;

The mixing ratio of reagent B and R is 2:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature, and the cells are observed after sedimentation for 2 minutes.

Comparative Example 2: (The difference between Comparative Example 2 and Example 2 is that the mixing ratio of liquid B and liquid R is different)

Reagent B composition and concentration: 0.1M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.1g/L new methylene blue, 0.3mL/L glutaraldehyde; 0.5% mannitol;

Reagent R composition and concentration: 0.1M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 7.0), 2g/L TritonX-100, 0.1g/L Eosin Y, 0.3mL/L glutaraldehyde; 0.5% mannitol;

The mixing ratio of reagent B and R is 1:2, and the ratio of the mixed solution to blood is 15:1; the sample is added to the counting plate and reacted at room temperature, and the cells are observed after sedimentation for 2 minutes.

Comparative Example 3: (Comparative Example 3 differs from Example 3 in that there is no mannitol)

Reagent B composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L TritonX-100, 0.3 g/L new methylene blue, 0.5 mL/L glutaraldehyde;

Reagent R composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L TritonX-100, 0.3 g/L Eosin Y, 0.5 mL/L glutaraldehyde;

The mixing ratio of reagent B and R is 4:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature and the cells are observed after sedimentation for 2 minutes.

Comparative Example 4: (The difference between Comparative Example 4 and Example 3 is that the types of anti-aggregating agents are different)

Reagent B composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L TritonX-100, 0.3 g/L new methylene blue, 0.5 mL/L glutaraldehyde, 0.012% EDTA-2Na;

Reagent R composition and concentration: 8 g/L NaCl, 0.01 M buffer composed of Na ₂ HPO ₄ and KH ₂ PO ₄ (pH 8.0), 6 g/L TritonX-100, 0.3 g/L Eosin Y, 0.5 mL/L glutaraldehyde, 0.012% EDTA-2Na;

The mixing ratio of reagent B and R is 4:1, and the ratio of the mixed solution to blood is 10:1; after the sample is added to the counting plate, it reacts at room temperature and the cells are observed after sedimentation for 2 minutes.

Comparative Example 5: (Refer to Example 1 of Patent Publication No. CN103398935A)

Reagent A composition and concentration: 5g/L NaCl, 0.02M buffer composed of Na ₂ HPO ₄ and NaH ₂ PO ₄ , 4mL/L hemolytic agent of octylphenyl polyoxyethylene ether, 0.1g/L KH ₂ PO ₄ , 0.15g/L methylene blue dye, 0.1mL/L glutaraldehyde stabilizer;

Reagent B composition and concentration: 5g/L NaCl, 0.02M buffer composed of Na ₂ HPO ₄ and NaH ₂ PO ₄ , 4mL/L hemolytic agent of octylphenyl polyoxyethylene ether, 0.1g/L KH ₂ PO ₄ , 0.5g/L eosin Y dye, 0.1mL/L glutaraldehyde stabilizer;

Reagent A and reagent B are mixed and added into the healthy blood sample to be tested (1 mL of reagent A, 1 mL of reagent B and 20 μL of blood sample).

Table 1 Comparison of dyeing effects of different embodiments

The results show that:

1. By comparing Example 1 with Comparative Example 1, it is shown that pH has a great influence on staining. The kit of the present invention has a better effect under a specific pH condition than under the acidic condition of pH 5.0 of Comparative Example 1. Under the specific pH condition, the leukocytes have a good morphology, no aggregation, and are clearly distinguished; while under the acidic condition of pH 5.0, the leukocytes have a good morphology, no aggregation, and a light color of eosinophils, which is not easy to distinguish from neutrophils (see Figure 2).

2. By comparing Example 2 with Comparative Example 2, the effect of the mixing ratio of reagent B and reagent R on staining is illustrated. When the mixing ratio of the two is between 2:1 and 4:1, the staining effect is best. As the content of reagent R increases, when the mixing ratio is 1:2, the neutrophil cytoplasm tends to be red, which is not very distinguishable from the deep red color of eosinophils, affecting the observation accuracy (see Figure 3).

3. By comparing Example 3 with Comparative Example 3, the role of mannitol in the present invention is illustrated. The experimental phenomena show that the use of mannitol enhances the cell staining efficiency and can effectively prevent leukocyte aggregation. It can be seen from Figure 4 that the use of a reagent without mannitol for staining results in lighter coloring of the leukocyte nuclei. It can be seen from Figure 5 that after staining with a reagent without mannitol, leukocytes aggregate to a certain extent, affecting the counting and test results.

4. By comparing Example 4 with Comparative Example 4, it is shown that if EDTA-2Na is used for anti-agglutination, the cell staining effect is better, but red blood cell fragments are aggregated and platelet and leukocytes are partially aggregated, which affects the instrument count.

5. By comparing Example 5 with Comparative Example 5, the staining effect of the technical solution of Patent Publication No. CN103398935A is not high in distinguishing the five types of white blood cells, and the cell nucleus is not obviously colored. The staining reagent used in this application shows better distinction of the five types of white blood cells, and the cell nucleus is obviously stained.

Experimental example: Accuracy comparison test

Reagent B and reagent R in Example 1-6 were used, mixed and added to the healthy blood sample to be tested (the mixing ratio of B to R was 2:1, and the ratio of the mixed solution to blood was 15:1), shaken and filled into the counting pool, incubated at room temperature for 2 minutes, and then sent to the analyzer for detection to obtain the five-classification white blood cell counting results. At the same time, the same blood sample was tested using a blood cell analyzer, and the results were compared, as shown in Table 2.

Table 2 Comparison of accuracy of the present invention

As shown in Table 2, there is no significant difference between the results of the leukocyte classification and counting using the kit of the present invention and the results of the electrical impedance blood cell analyzer commonly used in hospitals, and the proportions of various types of cells are at the same level, with high accuracy.

It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims (6)

1. A kit for differential counting of white blood cells, characterized in that: the reagent comprises a reagent B and a reagent R, wherein the components of the reagent B and the reagent R comprise dye, buffer solution, a penetrating agent, an osmotic pressure regulator, an anticoagulant, a preservative and a fixing agent;

1) The reagent B comprises the following components in percentage by weight:

dye selected from one of new methylene blue, brilliant tar blue, crystal violet, trypan blue, toluidine blue and allrin blue cation dye, with concentration of 0.1-0.5g/L;

buffer solution selected from HEPES, tris, MES, phosphate buffer solution and citric acid buffer solution, wherein the concentration is 0.01-0.10M, and the pH is 6.0-8.0;

A permeabilizer selected from one of NP40, saponin, tween 20, tween 40, brij35, brij52, brij58, brij72, triton X-100, triton X-114, triton X-165, and Triton X-305 nonionic surfactant at a concentration of 1-6g/L;

an osmotic pressure regulator selected from one of Na ₂SO₄, naCl and KCl, with concentration of 0-8g/L;

An anti-agglutinant selected from mannitol at a concentration of 0.3-0.7% v/v;

A fixing agent selected from one of formaldehyde, paraformaldehyde or glutaraldehyde, with a concentration of 0.1-0.5ml/L;

Preservative selected from one of sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, sodium azide, proClin300,300 or BND-10, and concentration of 0.1-0.5mL/L;

2) The reagent R comprises the following components in percentage by weight:

a dye selected from eosin Y and neutral red anion dye, wherein the concentration is 0.1-0.5g/L;

buffer solution selected from HEPES, tris-HCl, MES, phosphate buffer solution and citric acid buffer solution, wherein the concentration is 0.01-0.10M, and the pH is 6.0-8.0;

A permeabilizer selected from one of NP40, saponin, tween 20, tween 40, brij35, brij52, brij58, brij72, triton X-100, triton X-114, triton X-165, and Triton X-305 nonionic surfactant at a concentration of 1-6g/L;

an osmotic pressure regulator selected from one of Na ₂SO₄, naCl and KCl, with concentration of 0-8g/L;

An anti-agglutinant selected from mannitol at a concentration of 0.3-0.7% v/v;

A fixing agent selected from one of formaldehyde, paraformaldehyde or glutaraldehyde, with a concentration of 0.1-0.5ml/L;

Preservative selected from one of sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, sodium azide, proClin300,300 or BND-10, and concentration of 0.1-0.5mL/L;

3) The preparation method comprises the following steps: sequentially weighing dye, buffer solution, penetrating agent, osmotic pressure regulator, anticoagulant, fixative and antiseptic, placing in a beaker, adding ultrapure water to a set concentration, stirring until complete dissolution, and filtering to obtain the kit for classifying and counting leucocytes.

2. A kit for differential counting of white blood cells according to claim 1, wherein: reagent B:

the dye is selected from new methylene blue;

The buffer solution is selected from phosphate buffer solution consisting of Na ₂HPO₄、KH₂PO₄;

the penetrating agent is TritonX-100;

The anti-agglutinant is selected from mannitol;

the osmotic pressure regulator is selected from NaCl;

The fixing agent is selected from glutaraldehyde;

The preservative is selected from ProClin300,300.

3. A kit for differential counting of white blood cells according to claim 1, wherein: reagent Y:

the dye is selected from eosin Y;

The buffer solution is selected from phosphate buffer solution consisting of Na ₂HPO₄、KH₂PO₄;

the penetrating agent is TritonX-100;

The anti-agglutinant is selected from mannitol;

the osmotic pressure regulator is selected from NaCl;

The fixing agent is selected from glutaraldehyde;

The preservative is selected from ProClin300,300.

4. Use of a kit for differential counting of leukocytes according to any one of claims 1-3, wherein: is a method for differential counting of white blood cells, comprising the steps of:

1) Mixing the reagent B and the reagent R in the mixing ratio of (2-4): 1 to obtain a mixed dye liquor;

2) Adding a blood sample to be detected into the mixed dye liquor obtained in the step 1), wherein the ratio of the mixed dye liquor to the blood sample to be detected is (10-20), namely, 1, reacting at room temperature for instant coloring, shaking uniformly, immediately adding into a counting plate, and settling for 2min;

3) Sending the settled counting plate into an analyzer which is based on a machine vision principle and performs automatic identification and classification counting by an AI identification technology, and detecting to obtain a five-classification counting result of the white blood cells;

the specific morphological characteristics of the stained leukocytes are as follows:

① Neutrophil cytoplasm is colorless to faint yellow, and the nucleus is blue-purple, rod-shaped or split-leaf-shaped;

② Eosinophil cytoplasm stained orange to red, and nucleus stained blue-violet;

③ Basophil cytoplasm is purple red, and cell nucleus is blue-purple;

④ The lymphocyte and monocyte nucleus and cytoplasm are both stained blue, and the individual lymphocyte is small; large lymphocytes are larger than individual lymphocytes, but the cytoplasmic fraction is small and the nuclear staining is uniform; the nuclei of monocytes are horseshoe-shaped or rod-shaped.

5. The use of a kit for differential counting of leukocytes according to claim 4, wherein: the mixing ratio of the reagent B to the reagent R in the step 1) is 3:1.

6. The use of a kit for differential counting of leukocytes according to claim 4, wherein: the ratio of the mixed dye liquor to the blood sample to be tested in the step 2) is 10:1.