CN112362435A

CN112362435A - Cell staining reagent and cell staining method

Info

Publication number: CN112362435A
Application number: CN202011223029.8A
Authority: CN
Inventors: 王志平
Original assignee: Shenzhen Anlu Medical Technology Co ltd
Current assignee: Shenzhen Anlu Medical Technology Co ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-02-12

Abstract

A cell staining reagent and a method thereof, wherein the staining reagent comprises a first staining solution, a second staining solution and a diluent; the first dyeing liquid comprises a first dye, a second dye, an accelerating agent, a first accelerating agent and a first main solvent; the second staining solution comprises a second accelerating agent and a second main solvent. Uniformly mixing a sample to be dyed with a first dyeing solution and a diluent to finish primary dyeing; and adding a second dyeing solution for secondary dyeing after the primary dyeing. The reagent and the method can efficiently dye the active cells in the sample, have simple operation, good cell dyeing effect and obvious cell characteristics after dyeing, and are suitable for cell analysis under a bright field, such as cell morphology identification, cell classification identification and counting.

Description

Cell staining reagent and cell staining method

Technical Field

The application belongs to the technical field of cell staining, particularly relates to the technical field of cell staining before detection and analysis of cells in body fluid or secretion of organisms, and particularly relates to a method and a reagent for staining cells in body fluid or secretion.

Background

In the cellular detection of body fluids or secretions of living organisms, cell staining techniques are generally used to distinguish between different types, forms and states of cells.

In the conventional cell staining technique for brightfield observation and detection, a sample to be detected is usually smeared on a transparent carrier such as a glass slide, after the sample to be detected is dried, a dye solution and a buffer solution with different staining effects are sequentially dropped, after the dye solution and the buffer solution are uniformly mixed and dyed, a main body of the dye solution and the buffer solution influencing the observation is washed away, after the sample to be dyed is dried, the whole dyeing process is completed, and a stained sample smear for brightfield observation is formed.

FIG. 1 is a schematic diagram showing a process for preparing a blood smear in the prior art, which includes steps of preparing the blood smear and steps of staining the blood smear.

The step of making the blood smear comprises four small steps: respectively, step 1.1: dropping the blood sample onto the glass slide; step 1.2: taking another glass slide, inclining the glass slide according to an angle of 30-45 degrees, and pushing the glass slide to the other side at a constant speed; step 1.3: the blood film is evenly coated, and is in a flame shape to be a qualified smear; step 1.4: wait for the blood film to dry and prepare for staining.

The step of smear staining includes five small steps: respectively, step 2.1: marking with a pencil, flatly placing the blood smear on a dyeing rack, and marking with a crayon to prevent liquid from overflowing; step 2.2: naturally drying the blood smear, dripping 1-2 drops of dyeing liquid to cover the blood smear, and dyeing for about 1 minute; step 2.3: dropwise adding a little more buffer solution with the volume of 2-4 drops), and uniformly blowing the buffer solution and the dye solution by using an aurilave for about 5 minutes; step 2.4: gently shake the slide and then flush the stain from one side of the slide with a fine stream of tap water (note, need not pour the stain first and then flush) for about 1 minute; step 2.5: and stopping washing, and naturally drying the blood slices or sucking the blood slices by using filter paper to dry the blood slices, so that the blood slices can be used for microscopic examination.

As can be seen from the steps, the dyeing steps in the prior art are extremely complicated and time-consuming, the dyeing efficiency is extremely low, and the sample cell dyeing can not be carried out on a large scale. And the operation of each step needs to be trained by professional operators to prepare qualified observation samples.

The noun explains:

CAS Number (CAS Registry Number or CAS Rn, CAS #), also known as CAS accession Number or CAS accession Number, is a unique numerical identification Number for a substance (compound, polymeric material, Biological sequence, mixture, or alloy). It was constructed by the Chemical Abstracts Service (CAS) of the national institute of chemistry. The agency is responsible for assigning a CAS number to each substance appearing in the literature, avoiding the trouble of multiple names for chemical substances, and facilitating database retrieval.

The purified water is water meeting the standards of purified water in Chinese pharmacopoeia (2015 edition), and the main parameters are as follows: a pH of between 4.4 and 7.6, measured using an off-line conductivity meter, and a conductivity of no more than 25 μ S/cm (microsiemens per centimeter) of water at a temperature of 25 ℃ when the labeled loading is no more than 10 ml (milliliters).

Disclosure of Invention

The technical problem to be solved by the application is to avoid the defects in the prior art, provide a simple and efficient cell staining reagent and a method, and be suitable for large-scale and batch sample cell staining.

The technical scheme for solving the problems is that the cell staining reagent comprises a first staining solution and a diluent; the content range of the first dyeing liquid is 0.5-33% by volume percentage; the content range of the diluent is 67-99.5 percent by volume percentage; the pH value range of the cell staining reagent is 6-9.

The content of the first staining solution and the diluent ensures that the cell staining reagent stains cells in a proper active staining environment, so that the cell activity can be maintained for a longer time, and the cell activity is favorably utilized to enhance the staining effect. The dyeing reaction is carried out by utilizing the common Brownian motion of the active cells and the dye molecules in the solution, the dyeing speed is higher, and the dyeing efficiency is higher.

The cell staining reagent, wherein the first staining solution is independently encapsulated; independently packaging the diluent; when the reagent is used for staining cells, the first staining solution and the diluent are used in combination; the pH value range of the diluent is 6-9. The independently packaged first staining solution and the diluent can be respectively and independently sealed and stored, the respective characteristics of the first staining solution and the diluent are kept, and the first staining solution and the diluent are uniformly mixed during staining and then are jointly used to complete cell staining; the packaging mode is convenient to use after being prepared.

The first staining solution comprises: a first dye, a second dye, a first osmolality adjusting agent and a first main solvent; every 1000ml of first dyeing solution comprises 0.01g to 1.00g of first dye, 0.01g to 0.30g of second dye and 4.5g to 18g of first osmotic pressure regulator; the dosage of the first main solvent is determined by a constant volume method, namely the dosage of the first main solvent can enable other components to be dissolved to form the first dyeing solution with the volume of 1000 ml.

The volume or mass of the first main solvent is determined by a constant volume method. That is, the volume or mass of the first main solvent, the first dye, the second dye and the osmotic pressure regulator in the above amounts are dissolved in the first main solvent to form 1000ml of the first dyeing solution.

The first dye is cresyl violet and the second dye is benzidine; the first osmolality adjusting agent is sodium chloride; the first main solvent is purified water.

Each 1000ml of the first staining solution comprises 0.3g of cresol purple and 0.03g of benzidine.

The first dye and the second dye have dyeing groups selected to be especially suitable for living body cell dyeing, and the dyeing groups can penetrate cell membranes to enter cells by utilizing the active transport function of the cell membranes, so that the cell dyeing efficiency is further improved.

The first staining solution further comprises: an accelerating agent and a first accelerating agent; each 1000ml of the first dyeing solution comprises 1.05g to 31.50g of the dissolution accelerator and 0.22g to 1.08g of the first dyeing accelerator.

The cosolvent comprises anhydrous acetic acid or an acetic acid solution; the first accelerant comprises Tween-80 (Tween-80).

The cosolvent can be one or more of anhydrous acetic acid or acetic acid solution or anhydrous acetic acid. The acetic acid solution can be 98% acetic acid aqueous solution by volume percentage concentration; the acetic acid solution can also be an acetic acid aqueous solution with the mass percentage concentration of 98 percent; the acetic acid solution may also be replaced with anhydrous acetic acid. Each 1000ml of the first staining solution comprises 3.15g of acetic acid solution and 0.54g of first accelerating agent.

The first staining solution also comprises a preservative, and the preservative is 0.1-1 g in mass per 1000ml of the first staining solution.

The preservative comprises the combination of any one or more of propalin 300 (proclin-300), sodium azide and benzoate.

The amount of the preservative contained in the first dyeing solution was 0.5g per 1000 ml.

The cell staining reagent further comprises a second staining solution; the content range of the first dyeing liquid is 0.5-33% by volume percentage; the content range of the second dyeing solution in percentage by volume is 1-66%; the content range of the diluent is 1-97.5 percent by volume percentage.

The first staining solution is independently packaged; the diluent is independently packaged; the second staining solution is independently packaged; when the reagent is used for cell staining, the first staining solution, the second staining solution and the diluent are used in combination; the pH value range of the diluent is 6-9.

The second staining solution comprises: a second accelerant, a second osmolality adjusting agent, and a second main solvent; every 1000ml of the second dyeing solution comprises 33.3g to 1000g of the second accelerating agent, the mass of the second osmotic pressure regulator is 4.5g to 18g, and the dosage of the second main solvent is determined by a constant volume method, namely the dosage of the second main solvent can enable the components to form the second dyeing solution with the volume of 1000ml after being dissolved.

The second accelerant is an aqueous hydrogen peroxide solution; the second osmolality adjusting agent is sodium chloride; in the aqueous hydrogen peroxide solution, the mass percent concentration of hydrogen peroxide is 30%; the second main solvent is purified water.

The second staining solution can perform secondary staining on the living cells in the sample, so that the staining effect is better. And the second osmotic pressure regulator in the second staining solution enables the cell compatible characteristic of the second staining solution to be similar to that of the first staining solution, so that the active cells can have similar cell osmotic pressure environments when undergoing the first staining and the second staining, thereby better maintaining the cell activity during the staining process. The first staining solution, the second staining solution and the diluent which are independently packaged can be respectively and independently sealed and stored, the respective characteristics of the first staining solution, the second staining solution and the diluent are kept, and the first staining solution, the second staining solution and the diluent are uniformly mixed during staining and then are jointly used to complete cell staining; the packaging mode is convenient to use after being prepared.

Every 1000ml of the second dyeing solution comprises 33.3g to 333.3g of aqueous hydrogen peroxide; the mass of the aqueous hydrogen oxide solution may also be 116.55 g; the dosage of the purified water, namely the second main solvent, is determined by a constant volume method, namely the dosage of the second main solvent can enable the components to be dissolved to form a second dyeing solution with the volume of 1000 ml.

The first staining solution comprises: a first dye, a second dye, a first osmolality adjusting agent and a first main solvent; every 1000ml of first dyeing solution comprises 0.01g to 1.00g of first dye, 0.01g to 0.30g of second dye and 4.5g to 18g of first osmotic pressure regulator; the dosage of the first main solvent is determined by a constant volume method, namely the dosage of the first main solvent can dissolve other components to form a first dyeing solution with the volume of 1000 ml; the first dye is janus green B; the second dye is neutral red; the first tonicity modifier is sodium chloride; the first main solvent is purified water; the second staining solution comprises: the third dye is methyl blue; the mass of the third dye is 5g-25 g; the second accelerant is an aqueous hydrogen peroxide solution; the second osmolality adjusting agent is sodium chloride; in the aqueous hydrogen peroxide solution, the mass percent concentration of hydrogen peroxide is 30%; the second main solvent is purified water.

The first dye was janus green B, primarily used for staining of intracellular particulate matter; the second dye is neutral red, mainly used for staining cell nuclei; the third dye is methyl blue, primarily used for staining of the cytoplasm. The second staining solution not only carries out secondary staining through the second accelerating agent, but also carries out secondary staining on living cells in the sample through the third dye, and the staining effect is better due to the cooperation of the three dyes. And the second osmotic pressure regulator in the second staining solution enables the cell compatible characteristic of the second staining solution to be similar to that of the first staining solution, so that the active cells can have similar cell osmotic pressure environments when undergoing the first staining and the second staining, thereby better maintaining the cell activity during the staining process. The first staining solution, the second staining solution and the diluent which are independently packaged can be respectively and independently sealed and stored, the respective characteristics of the first staining solution, the second staining solution and the diluent are kept, and the first staining solution, the second staining solution and the diluent are uniformly mixed during staining and then are jointly used to complete cell staining; the packaging mode is convenient to use after being prepared.

The diluent comprises a buffering agent, a diluent osmotic pressure regulator and a diluent main solvent; the pH range of the diluent is 6.0-8.5. The buffer is used for acid-base equilibrium of the diluent; also known as acid-base balancing agents.

The diluent of each 1000ml comprises 0.121g to 12.1g of buffering agent; the mass of the diluent osmotic pressure regulator is 4.5g-18 g; diluting the main solvent to obtain purified water; the dosage of the main dilution solvent is determined by a constant volume method, namely the dosage of the main dilution solvent can ensure that the components can form a diluent with the volume of 1000ml after being dissolved.

The buffer includes: any one or more of Tris (hydroxymethyl) aminomethane (Tris), 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), Phosphate (PBS), 1, 4-piperazine diethylsulfonic acid (PIPES), N-carbamoylmethyl ethanesulfonic Acid (ACES) and 3-morpholine propanesulfonic acid (MOPS).

The diluent osmotic pressure regulator comprises: sodium chloride (NaCl), sodium sulfate (Na 2SO 4).

The diluent osmolality adjusting agent in the diluent allows the cell compatibility characteristics of the diluent to be similar to the cell compatibility characteristics of the first and second staining solutions, allowing the viable cells to have similar cell osmolality conditions when subjected to the dilution, first and second staining procedures, thereby better preserving cell viability during the staining procedure.

The technical solution for solving the above problems can also be a method for preparing a first staining solution, which is used for preparing the first staining solution; comprises the following steps of 1: weighing purified water, weighing cresyl violet, benzidine and sodium chloride, and uniformly mixing the four; step 2: filtering with 0.8 micrometer glass fiber filter membrane to obtain first dyeing solution. The time for mixing the above four ingredients uniformly is 24 hours.

The technical solution for solving the above problems can also be a method for preparing a first staining solution, which is used for preparing the first staining solution; comprises the following steps of 1: weighing purified water, and weighing Janus green B, neutral red and sodium chloride, and uniformly mixing the four materials; step 2: filtering with 0.8 micrometer glass fiber filter membrane to obtain first dyeing solution. The time for mixing the above four ingredients uniformly is 2 hours.

The technical solution for solving the above problems can also be a method for preparing a second dyeing solution, which is used for preparing the second dyeing solution; the method comprises the following steps: purified water is measured, 30% hydrogen peroxide water solution and sodium chloride are weighed and mixed evenly to prepare second dyeing solution. The time for mixing the three components evenly is 10 minutes.

The technical solution for solving the above problems can also be a method for preparing a second dyeing solution, which is used for preparing the second dyeing solution; the method comprises the following steps: purified water was measured, and 30% aqueous hydrogen peroxide, sodium chloride, and methyl blue were weighed and mixed to prepare a second staining solution. The time for mixing the above four uniformly was 15 minutes.

The technical scheme for solving the problems can also be a preparation method of the diluent, which is used for preparing the diluent; comprises the following steps of 1: weighing a dilution main solvent, weighing a buffer and a diluent osmotic pressure regulator, and uniformly mixing; step 2: adding an acidic substance or an alkaline substance into the diluent, and adjusting the pH value of the diluent to 6.0-8.5. The three are mixed evenly for 15 minutes. The acidic substance may be a conventional diluent-adjusting acid, such as hydrochloric acid or a hydrochloric acid solution; the alkaline substance may be a base, such as sodium hydroxide or sodium hydroxide solution, which is conventional for conditioning the dilution.

The technical solution of the present application for solving the above problems may also be a cell staining method using the above cell staining reagent; and uniformly mixing the sample to be dyed, the first dyeing solution and the diluent to finish the dyeing of the sample.

The technical scheme for solving the problems can also be a cell staining method, wherein the volume of a sample to be stained is 3-15 microliters; the volume ratio of a first comprehensive reagent volume formed by mixing the first staining solution and the diluent to a sample to be stained is 50: 1-300: 1; and in the process of finishing the dyeing by mixing the sample to be dyed and the first comprehensive reagent, the osmotic pressure range of cells in the mixed solution is 260 mmol/L-350 mmol/L.

According to the cell staining method, the volume ratio of the first comprehensive reagent formed by mixing the first staining solution and the diluent to the sample to be stained is within the range of 100: 1-200: 1; and in the process of mixing the sample to be dyed and the first comprehensive reagent to finish dyeing, the osmotic pressure range of cells in the mixed solution is 280-320 mmol/L.

When the sample to be dyed and the first comprehensive reagent are mixed to finish the dyeing process, the osmotic pressure range of cells in the mixed solution can also be 300 mmol/L; the ratio of the first integrated reagent volume to the volume of the sample to be stained may also range from 150: 1.

the technical solution of the present application for solving the above problems may also be a cell staining method using the above cell staining reagent; uniformly mixing a sample to be dyed, a first dyeing solution and a diluent to finish the first dyeing of the sample; and uniformly mixing the sample subjected to the first dyeing with the second dyeing solution, and carrying out secondary dyeing.

3-15 microliter of a sample to be dyed; the volume ratio of a second comprehensive reagent formed by mixing the first staining solution, the diluent and the second staining solution to a sample to be stained is in a range of 100: 1-300: 1; and in the process of mixing the sample to be dyed and the second comprehensive reagent to finish dyeing, the osmotic pressure range of cells in the mixed solution is 260 mmol/L-350 mmol/L.

The volume ratio of a second comprehensive reagent formed by mixing the first staining solution, the diluent and the second staining solution to a sample to be stained is 150: 1-280: 1; and in the process of mixing the sample to be dyed and the second comprehensive reagent to finish dyeing, the osmotic pressure range of the cells in the mixed solution is 290 mmol/L-310 mmol/L.

When the sample to be dyed and the second comprehensive reagent are mixed to finish the dyeing process, the osmotic pressure range of cells in the mixed solution can also be 300 mmol/L; the ratio of the volume of the second integrated reagent to the volume of the sample to be stained may also range from 200: 1 or 150: 1.

the technical solution of the present application for solving the above problems may also be a cell staining method using the above cell staining reagent; and comprises the following steps: step A1: diluting a sample to be dyed; adding a diluent into a sample to be dyed to prepare a sample diluent; the volume ratio range of sample and diluent is 1: 50-1: 300, respectively; step B1: first dyeing: taking the sample diluent in the step A1, adding the first staining solution, and uniformly mixing to prepare a sample solution after primary staining; the volume ratio of the sample dilution and the first staining solution ranges from 2: 1-194: 1; step C1: and (3) dyeing for the second time: taking the sample solution subjected to primary dyeing in the step B1, adding a second dyeing solution, and uniformly mixing to obtain a secondary dyeing sample solution; the volume ratio range of the sample liquid and the second staining liquid after the primary staining is 3: 2-49: 1.

the technical solution of the present application for solving the above problems may also be a cell staining method using the above cell staining reagent; and comprises the following steps: step A2: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1; step B2: preparing a comprehensive coloring agent diluent; taking the first dyeing solution diluent in the step A2, adding the second dyeing solution, and uniformly mixing to prepare a comprehensive dyeing agent diluent; the volume ratio range of the second staining solution and the first staining solution dilution is 3: 2-49: 1; step C2: b, dyeing the sample to be dyed, and adding the comprehensive dyeing agent diluent in the step B2 into the sample to be dyed; the ratio of the volume of the sample to be stained to the volume of the combined stain dilution liquids ranges from 1: 50-1: 300.

the technical solution of the present application for solving the above problems may also be a cell staining method using the above cell staining reagent; step A3: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1; step B3: b, dyeing the sample to be dyed, and adding the first dyeing solution diluent in the step A3 into the sample to be dyed to prepare a sample solution after primary dyeing; the volume ratio range of the sample to be dyed and the sample liquid after the primary dyeing is 1: 50-1: 300, respectively; step C3: preparing a secondary dyeing sample liquid; taking the sample solution subjected to primary dyeing in the step B3, adding a second dyeing solution, and uniformly mixing to obtain a secondary dyeing sample solution; the mass or volume ratio range of the second staining solution and the sample solution after the first staining is 3: 2-49: 1.

the cell staining method further comprises a step D: spreading the dyed sample; and C, taking any one of the secondary staining sample liquid obtained in the step C1, the step C2, the step C3 or the step C3, dripping the secondary staining sample liquid into a containing carrier, uniformly spreading the secondary staining sample liquid on the containing carrier, standing for 30 seconds to 1 minute, and obtaining the staining sample for bright field observation.

In the cell staining method, in the step a1, the step B1 and the step C1, in the step a2, the step B2 and the step C3, and in the step A3, the step B3 and the step C3, the mixing time is 20 seconds to 5 minutes or 30 seconds to 1 minute; the volume of the container for staining is not less than the amount of the cell staining reagent added, while the volume of the container for staining is not more than 1 time the amount of the reagent added.

The cell staining method further comprises the step of spreading the mixed solution after staining: dripping the dyed sample liquid into a containing carrier, and uniformly spreading the dyed sample liquid on the containing carrier; after standing for 30 seconds to 1 minute, a stained sample for bright field observation was obtained.

The holding carrier comprises a urine slagging counting plate, a cell counting plate, or an observation device or a glass slide made of transparent materials with a cavity.

The total staining time of the cell staining method is less than 8 minutes, or the total staining time interval ranges from 5 minutes to 6 minutes.

In the cell staining method, the sample to be stained comprises a blood sample, a urine sample and a leucorrhea sample; the blood sample comprises immediately collected peripheral venous blood or ethylenediamine tetraacetic acid (EDTA) anticoagulated venous blood; the volume of the sample to be stained is not less than 0.01 ml.

The environment temperature range of the cell staining method is 15-37 ℃.

Compared with the prior art, the beneficial effect of this application is: 1. the cells in the sample to be dyed are dyed in a liquid suspension, and the environment for the dyeing solution and the diluent is relatively close to the biological physiological state, so that the activity of the cells is better maintained in the dyeing process, the dyeing reaction can be carried out by utilizing the common Brownian motion of the cells and dye molecules in the solution and the electrostatic acting force, the dyeing speed is higher, and the efficiency is higher; 2. under the dyeing environment, the cell activity is kept better, and the dyeing liquid is particularly suitable for living cell dyeing through the selection of dyeing groups of dyeing materials in the dyeing liquid, and the dyeing groups can penetrate cell membranes to enter cells by utilizing the active transport function of the cell membranes, so that the cell dyeing efficiency is further improved; 3. the first staining solution, the second staining solution and the diluent which are independently packaged can be respectively and independently sealed and stored, the respective characteristics of the first staining solution, the second staining solution and the diluent are kept, and the first staining solution, the second staining solution and the diluent are uniformly mixed during staining and then are jointly used to complete cell staining; the packaging mode is convenient to prepare and use; 4. according to the method, the first staining solution, the second staining solution and the diluent have appropriate proportions of the components, so that the three solutions have high cell environment compatibility, and the cells can be kept active in the staining process; meanwhile, the three components can be flexibly prepared and used, and the method is suitable for dyeing different types of samples; 5. the second staining solution is adopted for secondary staining, the staining effect is better, the staining result is more beneficial to cell morphology and type identification, the stained solution can be directly suitable for cell morphology analysis and detection in a bright field, and cell classification counting can be further carried out according to the stained cell morphology and graphic feature analysis; 6. the cell staining can be completed in the liquid suspension of body fluid or secretion; the sample is suitable for various types of biological body fluids such as blood, secretion such as urine, leucorrhea and the like; 7. the dyeing operation is simple, and foolproof dyeing operation can be realized, so that the dyeing device is suitable for various application scenes. Such as point-of-care testing, emergency treatment, bedside, battlefield, etc., in a variety of conditions lacking professionals, large equipment and complex testing environments.

Drawings

FIG. 1 is a schematic representation of the steps of one embodiment of a cell staining method contemplated by the present application; the first staining solution in FIG. 1 is the first staining solution; the second staining solution in fig. 1 is the second staining solution;

FIG. 2 is a schematic diagram of a prior art blood smear making process;

figures 3 to 10 show pictures after blood cells have been stained; the images in fig. 3 to 10 are staining images formed by using the staining solutions prepared in the optimal amounts in tables 3 and 4 and using 0.01M phosphate buffer as a diluent according to the system and the procedure in any one of the examples;

FIG. 3 shows staining of neutrophils, the near center position of the image showing stained neutrophils; as seen near the center in fig. 3, the nucleus of neutrophils is blue, with branching structure, bluish cytoplasm, clean and no particulate matter;

FIG. 4 shows the staining of basophils, which is shown in the picture; as can be seen near the center of fig. 4, the basophilic nucleus region is bluish and covered by a large number of bluish-violet particles in the cytoplasm;

FIG. 5 shows the staining of eosinophils, and stained eosinophils are shown in FIG. 5; as seen at the box selection in FIG. 5, after eosinophils were stained, the cytoplasm was red with a large number of purple-red particles;

FIG. 6 shows the staining of lymphocytes, and FIG. 6 shows the stained lymphocytes; as can be seen in the boxed portion of fig. 6, the nuclei of lymphocytes are blue, large and round; few cytoplasmic areas, bluish;

FIG. 7 shows the staining of monocytes, the stained monocytes being shown in the picture; as can be seen in the near-center position of fig. 7, monocytes appear weakly basophilic, light blue cytoplasmic, bluish gray, without particulate matter; the cell nucleus is dark blue and irregular;

FIG. 8 shows the staining of platelets; as can be seen in the boxed portion of fig. 8, platelets are smaller than red blood cells, are purple-red spherical, and have purple-red particles in the cytoplasm;

FIG. 9 shows the condition where red blood cells are stained, and stained red blood cells are shown in FIG. 9; as can be seen in the boxed portion of fig. 9, the red blood cells: is purple red or blue purple, and is doughnut-shaped;

FIG. 10 shows the staining of reticulocytes, which are shown in the picture; reticulocytes are visible in the boxed portion of fig. 10: the red blood cells are stained with purple gray dotted and strip-shaped substances, and are doughnut-shaped.

Detailed Description

Embodiments of the present application will be described in further detail below with reference to the drawings.

The blood smear making steps include: step 1.1: dropping the blood sample onto the glass slide; step 1.2: taking another glass slide, inclining the glass slide according to an angle of 30-45 degrees, and pushing the glass slide to the other side at a constant speed; step 1.3: the blood film is evenly coated, and is in a flame shape to be a qualified smear; step 1.4: wait for the blood film to dry and prepare for staining.

The smear staining step comprises: step 2.1: marking with a pencil, flatly placing the blood smear on a dyeing rack, and marking with a crayon to prevent liquid from overflowing; step 2.2: after the blood smear is naturally dried, 1 to 2 drops of Rui-Ji's dye liquor (A liquor) are dripped to cover the blood smear for about 1 minute; step 2.3: dropping buffer solution (such as Ruizhi dye solution (B solution)) with a little more volume (2 drops to 4 drops), and uniformly blowing the buffer solution and the dye solution by using an aurilave for about 5 minutes; step 2.4: the slide is shaken slowly and then the stain is rinsed from one side of the slide with a fine stream of running water (without first pouring the stain and then rinsing) for about 1 minute; step 2.5: and stopping washing, and naturally drying the blood slices or sucking the blood slices by using filter paper to dry the blood slices, so that the blood slices can be used for microscopic examination.

As can be seen from the steps, the dyeing operation steps in the prior art are complex, the operation is complicated and time-consuming, the dyeing efficiency is extremely low, and the sample preparation of morphological bright field detection cannot be carried out on a large scale. And the operation of each step needs a trained professional operator to obtain a qualified sample for observing the dyed sample, and the operation requirement of the professional operator is high. The dyeing effect is often determined in the operation process, and the consistency of the dyeing quality is difficult to ensure; it is possible that the same operator may have a large difference in the final dyeing effect in different situations or different operators.

Further, in the staining operation step in the prior art, since the sample is subjected to the processes of smearing, spreading and drying the slide, most of the cells in the sample to be stained are "killed" during the staining process, and thus the staining only presents the state that most of the cells are inactivated, so that the stained smear cannot more truly reflect the physiological state of the cells in the body fluid.

In the examples shown in Table 1, the cell staining reagent is a first integrated reagent comprising only a first staining solution and a diluent; the formulation of some examples of the first staining solution is tabulated in table 1, with the ranges for the volume percentages of the first staining solution and the dilution being tabulated.

In the examples shown in Table 2, the cell staining reagent is a second integrated reagent comprising a first staining solution, a second staining solution and a diluent; the volume percent ranges for the first staining solution, the second staining solution and the dilution are listed in table 2.

In some embodiments of the cell staining method and reagent, the formulation and preparation method of the first staining solution, the formulation and preparation method of the second staining solution, and the formulation and preparation method of the diluent are included.

In an embodiment of the preparation of the first staining solution, the following steps are included:

the method comprises the following steps: weighing a certain amount of purified water, correspondingly weighing a proper amount of first dye, namely cresol purple, and mixing and uniformly mixing the purified water and the cresol purple.

Step two: weighing a proper amount of Tween-80 and a proper amount of proclin-300 as a first accelerant respectively, adding the solution prepared in the step one, and uniformly mixing for 60 minutes.

Step three: weighing a certain amount of second dye, namely benzidine, weighing a corresponding amount of 98% acetic acid, mixing the two, and uniformly mixing for 45 minutes.

Step four: and (4) mixing the mixed solution prepared in the step one and the mixed solution prepared in the step two in an equal volume ratio, and uniformly mixing for at least 24 hours.

Step five: and D, filtering the mixed solution prepared in the step four by using a glass fiber filtering membrane with the size of 0.8 micron, removing solid mechanical impurities in the solution, and obtaining the remaining liquid, namely the first dyeing liquid.

Table 3 is a table listing the formulation of the first dye solution section example. Table 4 is a table listing the formulations of the second dye solution section examples. Table 5 is a table listing the formulation makeup of some of the diluent examples.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

the method comprises the following steps: 995.48g of purified water and 0.3g of cresyl violet are weighed, mixed and mixed.

Step two: 0.54g of Tween-80 and 0.5g of proclin-300 are respectively weighed, the mixed solution prepared in the step one is added, and the mixture is uniformly mixed for 60 minutes.

Step three: 0.03g of benzidine, 3.15g of anhydrous acetic acid (3.15 g to 31.5 g) were weighed, mixed and mixed for 45 minutes.

Step four: and (4) mixing the mixed solution obtained in the second step and the mixed solution obtained in the third step in an equal volume ratio, and uniformly mixing for at least 24 hours.

Step five: and D, filtering the mixed solution obtained in the fourth step by using a glass fiber filtering membrane with the size of 0.8 micrometer to remove solid mechanical impurities in the solution, wherein the remained liquid part is the first dyeing solution.

the method comprises the following steps: dissolving 0.30g of cresol purple in 997.00ml of purified water to obtain a cresol purple aqueous solution; step two: then, 0.03g of benzidine was added to 3.00ml of acetic acid to obtain a benzidine acetic acid solution. The first step and the second step are not in sequence.

Step three: and mixing the cresol purple water solution with a benzidine acetic acid solution, adding 0.1ml of TritonX-100 (Triton-100), uniformly mixing, reacting for at least 24 hours, and filtering by using a 0.8-micron filter membrane to obtain the first staining solution.

the method comprises the following steps: weighing a certain amount of purified water, correspondingly weighing a proper amount of cresyl violet, mixing the purified water and the cresyl violet, and uniformly mixing.

Step two: weighing a proper amount of Tween-80 and a proper amount of proclin-300 as well as trihydroxymethyl aminomethane and sodium chloride as well as an accelerant respectively, adding the trihydroxymethyl aminomethane and the sodium chloride into the mixed solution prepared in the step 1, adjusting the pH value to 7.0, and uniformly mixing for 60 minutes.

Step three: weighing a certain amount of second dye benzidine, weighing a corresponding amount of 98% acetic acid, mixing the weighed two, and uniformly mixing for 45 minutes.

Step four: and (4) mixing the finished liquid in the second step and the liquid in the third step in an equal volume ratio, and uniformly mixing for at least 24 hours.

Step five: and D, filtering the mixed solution obtained in the fourth step by using a glass fiber filtering membrane with the size of 0.8 micrometer, removing solid mechanical impurities in the solution, and obtaining the remaining liquid, namely the dyeing liquid I.

The first staining solution has the main function of staining the micro-tissues in the cells of the body fluid, wherein the micro-tissues mainly comprise tissues such as cell nucleus, cytoplasm and other organelles. One of the dyes in the first dyeing solution is mainly formed by combining benzidine, especially at least one of cresol purple and rose red (tiger red), with micromolecule chemical dyes with colored groups and low cytotoxicity, such as neutral red, James green, toluidine blue, methylene blue, brilliant methylene blue, trypan blue, James green, cresol purple and rose red.

The dyeing liquid I mainly comprises a dye which is a micromolecular compound with a colored group, and further comprises buffer salt, osmotic pressure adjusting salt, a dissolution accelerator, a dyeing accelerator, an antiseptic and purified water; the small molecular compound with the color group is a compound with a color assisting group and a molecular weight of less than 500, and the main function of the small molecular compound is to enable components in cells to be colored so as to facilitate subsequent morphological observation and classification.

The first dye cresol purple, having CAS number 10510-54-0, molecular formula C18H15N3O3, molecular weight 321.33, has a mass concentration in the bulk dye system in the range of 0.01 g/L to 1g/L, preferably 0.3 g/L.

The second dye is 3,3' -dimethylbenzidine having a CAS number of 626-13-1, a molecular formula of C14H15N, a molecular weight of 197.28, and a mass concentration in the bulk dye system in the range of 0.01g to 0.3g/L, preferably 0.03 g/L.

In an embodiment of the preparation of the second staining solution, the following steps are included:

the method comprises the following steps: 883.45g of purified water is weighed, 116.55g of hydrogen peroxide solution with the mass fraction of 30% is weighed, and the mixture is stirred for 10 minutes to be uniformly mixed; this is the second staining solution.

The second staining solution has the main function that after the blood sample is mixed with the blood sample diluent and the first staining solution, the second staining solution is added to characteristically stain peroxidase particles in certain white blood cells in the blood cells; the main component in the second dyeing solution is aqueous hydrogen peroxide solution with a certain mass ratio.

One embodiment of the diluent comprises: trihydroxymethyl aminomethane, sodium chloride, and purified water are added, mixed, and added with appropriate amount of acidic substance or alkaline substance, pH value is adjusted to 7.4, and volume is adjusted to 1000 ml. Mixing the components in the formula of the diluent, fully dissolving, adjusting the pH value of the solution to 7.4, metering to 1000ml, and filtering and sterilizing by using a 0.22 micron glass fiber filter membrane to obtain the diluent.

The main function of the diluent includes several aspects, one of which is to dilute the blood cells in a certain proportion so that after staining with the method and the reagent, the blood cells can be observed under a microscope in a proper proportion; secondly, a liquid pH environment suitable for a blood cell staining reaction is provided; thirdly, providing an osmotic pressure environment for ensuring the normal morphology of blood cells in the sample; the diluent can be a common buffer system for biological experiments. The method comprises the following steps: the main function of the buffer salt in the dilution is to provide a suitable pH environment for the staining reaction of the blood cells. The pH value range of the diluent is 5-11, the relatively preferable range of the pH value of the diluent is 6.0-8.5, and the particularly preferable range of the pH value of the diluent is 6.8-7.4; the mixed solution composed of the diluent, the first staining solution and the second staining solution has a range of 150mmol/L to 400mmol/L, preferably 260mmol/L to 350mmol/L, relative to the cell osmotic pressure environment. mmol/L is millimoles per liter.

Purifying water, namely a main solvent in the first dyeing solution, the second dyeing solution and the diluent, and dissolving various components; the purified water may also be replaced with deionized or distilled water as desired.

The cosolvent mainly has the function of promoting the dye molecules to be fully dissolved in a dyeing liquid system. The main role of the accelerating agent is to allow intracellular components to stain with greater efficiency. The main function of the preservative is to prevent the growth of other microorganisms in the dye liquor.

Fresh samples to be stained, such as blood samples, have cells therein that are biologically active. Adding a low-cytotoxicity staining reagent in a solution environment with a proper pH value and a proper cell osmotic pressure close to a physiological state; because of the biological activity of blood cells, the small molecule dye is phagocytized into the cells by utilizing the endocytosis of the blood cells; the dyes endocytosed into the cell belong to the polychrome class of dyes, which in the intracellular environment, due to different group charge properties, determine that different dye molecules bind to different substances within the cell and give the intracellular substances different colours. In addition, the second staining solution of the method can further specifically promote the components in the first staining solution to react with peroxidase particles in cells and color the peroxidase particles.

In one embodiment, the cell staining method comprises the following steps:

step A1: diluting a sample to be dyed; adding a diluent into a sample to be dyed; the volume ratio range of sample and diluent is 1: 50-1: 300, respectively; the sample to be stained is an ex vivo sample with viable cells.

Step B1: dyeing a sample to be dyed for the first time; taking the sample diluent in the step A1, adding the first staining solution, and uniformly mixing to prepare a sample solution after primary staining; the sample dilution and first staining solution volume ratio ranges from 2: 1-194: 1.

step C1: dyeing the sample to be dyed for the second time; taking the sample solution subjected to primary dyeing in the step B1, adding a second dyeing solution, and uniformly mixing to obtain a secondary dyeing sample solution; the volume ratio range of the sample liquid and the second staining liquid after the primary staining is 3: 2-49: 1.

in one embodiment, the cell staining method comprises the following steps:

step A2: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1.

step B2: preparing a comprehensive coloring agent diluent; taking the first dyeing solution diluent in the step A2, adding the second dyeing solution, and uniformly mixing to prepare a comprehensive dyeing agent diluent; the volume ratio range of the second staining solution and the first staining solution dilution is 3: 2-49: 1.

step C2: b, dyeing the sample to be dyed, and adding the comprehensive dyeing agent diluent in the step B2 into the sample to be dyed; the ratio of the volume of the sample to be stained to the volume of the combined stain dilution liquids ranges from 1: 50-1: 300.

in one embodiment, the cell staining method comprises the following steps:

step A3: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1.

step B3: b, dyeing the sample to be dyed, and adding the first dyeing solution diluent in the step A3 into the sample to be dyed to prepare a sample solution after primary dyeing; the volume ratio range of the sample to be dyed and the sample liquid after the primary dyeing is 1: 50-1: 300.

step C3: preparing a secondary dyeing sample liquid; taking the sample solution subjected to primary dyeing in the step B3, adding a second dyeing solution, and uniformly mixing to obtain a secondary dyeing sample solution; the mass or volume ratio range of the second staining solution and the sample solution after the first staining is 3: 2-49: 1.

the dyeing method of each cell also comprises the step of spreading the sample mixed solution after dyeing; and C, taking any secondary dyeing sample liquid obtained in the steps C1-C3, uniformly spreading the secondary dyeing sample liquid on the containing carrier, covering the covering sheet, and standing for 30 seconds to 1 minute to obtain the dyeing sample for bright field observation.

In the step A1, the step B1 and the step C1, in the step A2, the step B2 and the step C3, and in the step A3, the step B3 and the step C3, the mixing time is 20 seconds to 5 minutes, or 30 seconds; the components of the reagent are mixed in a container with the volume not less than the volume of the added amount of the liquid, and the volume of the container is not less than the volume of the added amount of the reagent and not more than 1 time of the volume of the added amount of the reagent.

In the above cell staining methods, the sample to be stained includes a blood sample, a urine sample, a leucorrhea sample, and a sputum sample; the blood sample comprises immediately collected peripheral venous blood or ethylenediamine tetraacetic acid (EDTA) anticoagulated venous blood; the volume is not less than 0.01 ml.

The single-step dyeing time of each dyeing step is 30 seconds to 1 minute; the combined comprehensive dyeing time of the dyeing steps is less than 8 minutes, and the optimal comprehensive dyeing time interval range is 5-6 minutes.

The environment temperature range of dyeing in the dyeing method is 25-37 ℃.

In some embodiments of the cell staining method and reagent, the sample to be stained is not less than 10ul of whole fingertip blood, or ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: dilution of blood samples: with 0.9% sodium chloride (NaCl) solution (normal saline), or 0.01M phosphate buffer (PBS buffer) at pH 6.8, 0.01M means 0.01 mol per liter, at 1: 50 to 1: diluting the blood sample within 300 times, and blowing and beating the blood sample by using a pipette for 30 seconds to mix the blood sample uniformly, or reversing the blood sample for 1 minute to mix the blood sample uniformly; that is, a pipette or other quantitative blood sampling device is used to take 10ul of whole blood sample, add it into a diluent containing 2ml of physiological saline or phosphate buffer solution (PBS buffer solution), and mix well.

Step two: first dyeing: and (3) adding 5ul of the first staining solution into the sample diluent prepared in the step one, and blowing and beating the mixture by using a pipette for 30 seconds to mix the mixture evenly, or reversing the mixture for 1 minute to mix the mixture evenly.

Step three: and (3) dyeing for the second time: and (3) adding 10ul of second staining solution into the sample mixed solution obtained after the first staining in the step two, blowing and beating the mixture by using a pipette for 30 seconds, and uniformly mixing the mixture or reversing the mixture for 1 minute.

Step four: and (4) taking one glass slide, dripping 10ul of the sample mixed solution obtained in the third step after the second dyeing on the glass slide, covering a cover glass, and standing for 1 minute.

Step five: the slide glass is taken and placed under a common optical microscope, and morphological observation and counting of Red Blood Cells (RBC), White Blood Cells (WBC) and Platelets (PLT) can be carried out.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a2 ml centrifuge tube is taken, 10ul of whole blood sample is taken by a quantitative blood sampling glass tube or a pipette, 1975ul of normal saline or 0.01M phosphate buffer solution is added, and the mixture is blown by a 1ml pipette for 30 seconds and is mixed evenly.

Step two: and (3) taking 5ul of the first staining solution by using a 10ul pipette, adding the first staining solution into the mixed solution obtained by diluting the sample in the step one, and blowing and beating the mixed solution by using the pipette for 30 seconds to uniformly mix the mixed solution to obtain the mixed solution obtained after the first staining.

Step three: and (3) taking 10ul of the second staining solution by using a 10ul pipette, adding the second staining solution into the mixed solution obtained after the first staining in the step two, and blowing and beating the mixed solution by using the pipette for 30 seconds to uniformly mix the mixed solution.

Step four: and (4) taking one glass slide, dripping 10ul of the mixed solution obtained in the third step after the second dyeing on the glass slide, covering a cover glass, and standing for 1 minute.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a2 ml centrifuge tube was taken and 1975ul of physiological saline or 0.01M phosphate buffer was added.

Step two: and (3) taking 5ul of the first staining solution by using a 10ul pipette, adding the first staining solution into the mixed solution prepared in the step one, and blowing and beating the first staining solution by using the pipette for 30 seconds to mix the first staining solution and the second staining solution uniformly.

Step three: and (3) taking 10ul of the second staining solution by using a 10ul pipette, adding the second staining solution into the mixed solution prepared in the step (II), and blowing and beating the second staining solution by using the pipette for 30 seconds to mix the second staining solution uniformly.

Step four: and (3) taking 10ul of whole blood sample by using a quantitative blood sampling glass tube or a pipette, adding the whole blood sample into the mixed solution prepared in the step three, and blowing and beating the whole blood sample by using the pipette for 30 seconds to uniformly mix the whole blood sample.

Step five: and (4) taking one glass slide, dripping 10ul of the mixed solution obtained in the fourth step after the second dyeing on the glass slide, covering a cover glass, and standing for 1 minute.

Step six: the slide glass is taken and placed under a common optical microscope, and morphological observation and counting of Red Blood Cells (RBC), White Blood Cells (WBC) and Platelets (PLT) can be carried out.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a2 ml centrifuge tube was taken and 1975ul of physiological saline or 0.01M PBS (phosphate buffered saline) was added thereto.

Step two: and taking 5ul of the first staining solution by using a 10ul pipette, adding the first staining solution into the mixed solution obtained in the step one, and blowing the first staining solution by using the pipette for 30 seconds to mix the first staining solution and the second staining solution uniformly.

Step three: and (3) taking 10ul of whole blood sample by using a quantitative blood sampling glass tube or a pipette, adding the whole blood sample into the mixed solution prepared in the step two, and blowing and beating the whole blood sample by using the pipette for 30 seconds to uniformly mix the whole blood sample.

Step four: and (4) taking 10ul of the second staining solution by using a 10ul pipette, adding the second staining solution into the mixed solution prepared in the third step, and blowing and beating the second staining solution by using the pipette for 30 seconds to mix the second staining solution uniformly.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a2 ml centrifuge tube was taken and 975ul of physiological saline or 0.01M PBS (phosphate buffered saline) was added.

Step five: and adding 1000ul of normal saline or 0.01M PBS (phosphate buffer solution) into the mixed solution prepared in the fourth step, and blowing and beating the mixed solution by a pipette to be uniformly mixed, or manually reversing the mixed solution for 30 seconds to be uniformly mixed.

Step six: and (4) taking one glass slide, dripping 10ul of the mixed solution obtained in the fifth step after the second dyeing on the glass slide, covering a cover glass, and standing for 1 minute.

Step seven: the slide glass is taken and placed under a common optical microscope, and morphological observation and counting of Red Blood Cells (RBC), White Blood Cells (WBC) and Platelets (PLT) can be carried out.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a1.5 ml centrifuge tube is taken, 5ul of whole blood sample is taken by a quantitative blood sampling glass tube or a pipette, 970ul of diluent is added, and the mixture is blown by a 1ml pipette for 30 seconds and mixed evenly.

Step two: and (3) adding 5ul of the first staining solution into the mixed solution prepared in the step one, and blowing and beating the mixed solution by using a pipette for 1 minute to mix the mixed solution uniformly.

Step three: and (3) adding 20ul of the second staining solution into the mixed solution prepared in the step two, and blowing and beating the mixed solution by using a pipette for 1 minute to mix the mixed solution uniformly.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a1.5 ml centrifuge tube was filled with 970ul of diluent.

Step four: and (3) taking 5ul of whole blood sample by using a quantitative blood sampling glass tube or a pipette, adding the whole blood sample into the mixed solution prepared in the step two, and blowing and beating the whole blood sample by using the pipette for 1 minute to mix the whole blood sample uniformly.

In some embodiments of the cell staining methods and reagents, the steps are:

Step two: taking 5ul of first staining solution, adding the first staining solution into the mixed solution prepared in the step one, and blowing and beating the first staining solution by using a pipettor for 1 minute and uniformly mixing the first staining solution and the mixed solution;

step three: and (3) taking 5ul of whole blood sample by using a quantitative blood sampling glass tube or a pipette, adding the whole blood sample into the solution prepared in the step two, and blowing and beating the whole blood sample by using the pipette for 1 minute to mix the whole blood sample evenly.

Step four: and (5) adding 10ul of second staining solution into the mixed solution prepared in the third step, and blowing and beating the mixed solution by using a pipette for 1 minute to mix the mixed solution uniformly.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: a1.5 ml centrifuge tube was taken and 470ul of diluent was added.

Step three: and (3) taking 5ul of whole blood sample by using a quantitative blood sampling glass tube or a pipette, adding the whole blood sample into the mixed solution prepared in the step two, and blowing and beating the whole blood sample by using the pipette for 1 minute to mix the whole blood sample uniformly.

Step four: and (4) adding 10ul of second staining solution into the mixed solution prepared in the third step, and blowing and beating the mixed solution by using a pipette for 1 minute to mix the mixed solution uniformly.

Step five: and adding 500ul of diluent into the solution prepared in the step four, and uniformly mixing by blowing with a pipette or manually reversing for 1 minute.

In some embodiments of the cell staining methods and reagents, the steps are:

Step four: and (4) taking one glass slide, dripping 10ul of the mixed solution prepared in the step three on the glass slide, covering the glass slide, and standing for 1 minute.

In each of the above embodiments, the slide may include a urine sediment counting plate, or a cell counting plate, or other viewing device made of a transparent material with a cavity.

In some embodiments of the cell staining methods and reagents, the steps are:

the method comprises the following steps: and (3) sampling by using a quantitative sampling device, wherein the sampling amount is not less than 10 microliter.

Step two: diluting and mixing the sample, wherein the dilution ratio is that: diluent = 1: 10 to 1: 1000, parts by weight; preferably 1: 100 to 1: 300, recommendation 1: 200 of a carrier; the mixing process can be uniformly mixed in any power mode, the mixing time is 20 seconds to 5 minutes, and the preferable mixing time is 30 seconds; the above-mentioned mixing agents may be mixed in a container not less than the amount of the liquid to be added. The volume of the container is not less than the volume of the added amount of the reagent and not more than 1 time of the volume of the added amount of the reagent.

Step three: the first dyeing, adding the first dyeing solution into the mixed solution prepared in the second step, and dyeing the cells in the mixed solution; after the first staining solution is added, the first staining solution can be mixed uniformly by any power mode, the mixing time is 20 seconds to 5 minutes, and the preferred mixing time is 1 minute.

Step four: and (3) second dyeing, namely adding a second dyeing solution into the mixed solution prepared in the third step to perform secondary dyeing on the cells, and uniformly mixing the cells in any power mode for 20 seconds to 5 minutes, wherein the preferable mixing time is 1 minute.

Step five: and (3) loading and tiling the sample mixed solution, namely adding the mixed solution prepared in the fourth step on a transparent observation device capable of forming a certain cavity, wherein the adding amount is 3 microliters to 50 microliters, preferably 8-15 microliters, and most preferably 10 microliters.

Step six: and a step of standing the sample mixture, in which the transparent observation device is moved to a position under a microscope or other observation devices, and is horizontally left standing for 20 seconds to 5 minutes, preferably 1 minute.

In the cell staining reagent and method of the present application, the technical effects thereof include various aspects.

First, a fresh sample of body fluid or secretions, the cells within which are biologically active. Adding a staining reagent with low cytotoxicity under a proper buffer system which is close to the physiological state (pH value and osmotic pressure); such an environment is closer to the original physiological environment of the cell, and the cell activity is maintained for a longer period of time.

Secondly, under the environment, the biological activity of cells in the sample is well maintained, and the small molecular dye can be phagocytized into the cells by utilizing the endocytosis of the cells, namely the active phagocytic function of active cells is utilized for dyeing.

Thirdly, the dye endocytosed into the cell belongs to multicolor dye, and in the intracellular environment, due to different group charge attributes, the attributes determine that different dye molecules are combined with different substances in the cell, and the substances in the cell are provided with different colors.

Fourthly, the second staining solution of the method can further specifically promote the components in the first staining solution to react with the peroxidase particles in the cells and color the peroxidase particles.

The cell staining reagent and the staining method can complete cell staining in liquid suspension of body fluid or secretion. The sample suitable for the method comprises various biological body fluids such as blood, secretion such as urine or leucorrhea and the like.

According to the cell staining method, cells in a sample to be stained finish a staining process in a liquid suspension, and the environments for a staining solution and a diluent are relatively close to the biological physiological state, so that the cell activity is better maintained in the staining process, on one hand, the common Brownian motion of the cells and dye molecules in the solution and the electrostatic acting force can be utilized to perform a staining reaction, the staining speed is higher, and the efficiency is higher; on the other hand, under the dyeing environment, the cell activity is better maintained, and the dyeing group selection of the dyeing material in the dyeing solution enables the dyeing material to penetrate through cell membranes to enter cells by utilizing the active transport function of the cell membranes, so that the dyeing method is particularly suitable for dyeing living cells, and further improves the cell dyeing efficiency.

According to the method, the ratio of the sample dyeing reagent to the sample is set reasonably, living cells in the sample can be dyed, the operation is simple, the dyeing speed is high, the dyeing effect is better, the dyeing result is more beneficial to cell morphology and type identification, the dyed solution can be directly suitable for cell morphology analysis and detection in a bright field, and cell classification counting can be further carried out according to the cell morphology and graphic feature analysis after dyeing.

In the field of medical application, it is not seen that in vitro vital staining is performed on a blood sample in a state that cells are in a solution state, that is, a compound dye with a colored group is adopted to directly stain blood cells, and cell classification analysis is performed according to cell characteristics presented by the staining results of the blood cells under a bright field microscope. The body fluid or secretion stained by the method of the present application presents cellular features including the size, color, morphology, shape of nucleus, color, cytoplasm color, and size and amount of particulate matter in cytoplasm. The stained sample in the present application is suitable for morphological analysis of cells, and classification and counting of cells can be performed based on analysis of cell characteristics such as cell morphology and staining degree.

Compared with the dyeing method and reagent in the prior art, the method and the reagent have the main characteristics that when the method and the reagent are used for dyeing, the state of the sample cells is in a physiological or near-physiological state, namely, the sample cells have vital activity in a physiological saline or physiological salt concentration state; whereas the stained sample of the prior art is typically mounted on a transparent carrier such as a glass slide, the cells are typically dead cells in a "dry" state.

Compared with the dyeing method and reagent in the prior art, the method has the main characteristics that the reaction environment and the operation are completely different during dyeing, and the drying and cleaning processes are not involved in the method; in this application, the cell in the sample is in liquid solution environment always, under no extra operation flow, according to the common brownian motion of dye molecule in blood cell and the solution, and the static acting force, carries out the dyeing reaction, and dyeing efficiency is higher.

The cell staining reagent and the cell staining method have the advantages of simple operation, low requirement on operators, uniform staining and short time consumption, and can realize a foolproof staining process; the defects of complex cell dyeing operation, high professional requirement, long time consumption, uneven dyeing and high dyeing material cost in the prior art are overcome. The dyeing operation is simple, and foolproof dyeing operation can be realized, so that the dyeing device is suitable for various application scenes. The scene such as the instant check is also suitable for a plurality of application scenes such as emergency treatment, bedside, battlefield and the like which lack large professional equipment, professionals and complex check environments.

The above description is only an example of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the present specification and the accompanying drawings, or which are directly or indirectly applied to other related technical fields, are intended to be included within the scope of the present application.

Claims

1. A cell staining reagent characterized in that,

comprises a first staining solution and a diluent;

the content range of the first dyeing liquid is 0.5-33% by volume percentage;

the content range of the diluent is 67-99.5 percent by volume percentage;

the pH value range of the cell staining reagent is 6-9.

2. The reagent for cell staining according to claim 1,

the first staining solution is independently packaged; the diluent is independently packaged;

when the reagent is used for staining cells, the first staining solution and the diluent are used in combination;

the pH value range of the diluent is 6-9.

3. The cell staining reagent according to claim 1 or 2,

the first staining solution comprises: a first dye, a second dye, a first osmolality adjusting agent and a first main solvent;

every 1000ml of first dyeing solution comprises 0.01g to 1.00g of first dye, 0.01g to 0.30g of second dye and 4.5g to 18g of first osmotic pressure regulator; the dosage of the first main solvent is determined by a constant volume method, namely the dosage of the first main solvent can enable other components to be dissolved to form the first dyeing solution with the volume of 1000 ml.

4. The cell staining reagent according to claim 3,

5. The cell staining reagent according to claim 4,

6. The cell staining reagent according to claim 3,

the first staining solution further comprises: an accelerating agent and a first accelerating agent;

each 1000ml of the first dyeing solution comprises 1.05g to 31.50g of the dissolution accelerator and 0.22g to 1.08g of the first dyeing accelerator.

7. The cell staining reagent according to claim 6,

the cosolvent comprises anhydrous acetic acid or an acetic acid solution;

the first accelerant comprises Tween-80 (Tween-80).

8. The cell staining reagent according to claim 3,

9. The reagent for cell staining according to claim 8,

10. The cell staining reagent according to claim 1 or 2,

the dyeing liquid also comprises a second dyeing liquid;

the content range of the first dyeing liquid is 0.5-33% by volume percentage;

the content range of the second dyeing solution in percentage by volume is 1-66%;

the content range of the diluent is 1-97.5 percent by volume percentage.

11. The reagent for cell staining according to claim 10,

the first staining solution is independently packaged;

the diluent is independently packaged;

the second staining solution is independently packaged;

when the reagent is used for cell staining, the first staining solution, the second staining solution and the diluent are used in combination;

the pH value range of the diluent is 6-9.

12. The reagent for cell staining according to claim 11,

the second staining solution comprises: a second accelerant, a second osmolality adjusting agent, and a second main solvent;

every 1000ml of the second dyeing solution comprises 33.3g to 1000g of the second accelerating agent, the mass of the second osmotic pressure regulator is 4.5g to 18g, and the dosage of the second main solvent is determined by a constant volume method, namely the dosage of the second main solvent can enable the components to form the second dyeing solution with the volume of 1000ml after being dissolved.

13. The reagent for cell staining according to claim 12,

the second accelerant is an aqueous hydrogen peroxide solution; the second osmolality adjusting agent is sodium chloride;

in the aqueous hydrogen peroxide solution, the mass percent concentration of hydrogen peroxide is 30%; the second main solvent is purified water.

14. The reagent for cell staining according to claim 12,

every 1000ml of first dyeing solution comprises 0.01g to 1.00g of first dye, 0.01g to 0.30g of second dye and 4.5g to 18g of first osmotic pressure regulator; the dosage of the first main solvent is determined by a constant volume method, namely the dosage of the first main solvent can dissolve other components to form a first dyeing solution with the volume of 1000 ml;

the first dye is janus green B; the second dye is neutral red; the first tonicity modifier is sodium chloride; the first main solvent is purified water;

the second staining solution comprises: the third dye is methyl blue; the mass of the third dye is 5g-25 g; the second accelerant is an aqueous hydrogen peroxide solution; the second osmolality adjusting agent is sodium chloride; in the aqueous hydrogen peroxide solution, the mass percent concentration of hydrogen peroxide is 30%; the second main solvent is purified water.

15. The cell staining reagent according to claim 1 or 2,

the diluent comprises a buffering agent, a diluent osmotic pressure regulator and a diluent main solvent;

the pH range of the diluent is 6.0-8.5.

16. The reagent for cell staining according to claim 15,

the diluent of each 1000ml comprises 0.121g to 12.1g of buffering agent;

the mass of the diluent osmotic pressure regulator is 4.5g-18g, and the main diluent solvent is purified water; the dosage of the main dilution solvent is determined by a constant volume method, namely the dosage of the main dilution solvent can ensure that the components can form a diluent with the volume of 1000ml after being dissolved.

17. The reagent for cell staining according to claim 15,

18. The reagent for cell staining according to claim 15,

19. A method for preparing a first staining solution, which is characterized in that,

for preparing a first staining solution according to claim 4;

comprises the following steps of 1: weighing purified water, weighing cresyl violet, benzidine and sodium chloride, and uniformly mixing the four;

step 2: filtering with 0.8 micrometer glass fiber filter membrane to obtain first dyeing solution.

20. A method for preparing a first staining solution, which is characterized in that,

for preparing a first staining solution according to claim 14;

comprises the following steps of 1: weighing purified water, and weighing Janus green B, neutral red and sodium chloride, and uniformly mixing the four materials;

21. A method for preparing a second dyeing liquid is characterized in that,

for preparing a second staining solution according to claim 13;

the method comprises the following steps: weighing purified water, weighing 30% hydrogen peroxide water solution and sodium chloride, and uniformly mixing the purified water, the 30% hydrogen peroxide water solution and the sodium chloride; and preparing a second dyeing solution.

22. A method for preparing a second dyeing liquid is characterized in that,

for preparing a second staining solution according to claim 14;

the method comprises the following steps: weighing purified water, weighing 30% aqueous hydrogen peroxide, sodium chloride and methyl blue, and mixing the four; and preparing a second dyeing solution.

23. A method for preparing a diluent is characterized in that,

for preparing the dilution of claim 15;

comprises the following steps of 1: weighing a dilution main solvent, weighing a buffer and a diluent osmotic pressure regulator, and uniformly mixing;

step 2: adding an acidic substance or an alkaline substance into the diluent, and adjusting the pH value of the diluent to 6.0-8.5.

24. A method for staining cells, characterized in that,

using the cell staining reagent of claim 1;

and uniformly mixing the sample to be dyed, the first dyeing solution and the diluent to finish the dyeing of the sample.

25. The cell staining method according to claim 24,

3-15 microliter of a sample to be dyed;

the volume ratio of a first comprehensive reagent volume formed by mixing the first staining solution and the diluent to a sample to be stained is 50: 1-300: 1;

and in the process of finishing the dyeing by mixing the sample to be dyed and the first comprehensive reagent, the osmotic pressure range of cells in the mixed solution is 260 mmol/L-350 mmol/L.

26. The cell staining method according to claim 25,

the volume ratio of a first comprehensive reagent volume formed by mixing the first staining solution and the diluent to a sample to be stained is 100: 1-200: 1;

and in the process of finishing the dyeing by mixing the sample to be dyed and the first comprehensive reagent, the osmotic pressure range of the cells in the mixed solution is 280 mmol/L-320 mmol/L.

27. A method for staining cells, characterized in that,

using the cell staining reagent of claim 12;

uniformly mixing a sample to be dyed, a first dyeing solution and a diluent to finish the first dyeing of the sample;

and uniformly mixing the sample subjected to the first dyeing with the second dyeing solution, and carrying out secondary dyeing.

28. The cell staining method according to claim 27,

3-15 microliter of a sample to be dyed;

the volume ratio of a second comprehensive reagent formed by mixing the first staining solution, the diluent and the second staining solution to a sample to be stained is in a range of 100: 1-300: 1;

and in the process of mixing the sample to be dyed and the second comprehensive reagent to finish dyeing, the osmotic pressure range of cells in the mixed solution is 260 mmol/L-350 mmol/L.

29. The cell staining method according to claim 28,

the volume ratio of a second comprehensive reagent formed by mixing the first staining solution, the diluent and the second staining solution to a sample to be stained is 150: 1-280: 1;

and in the process of mixing the sample to be dyed and the second comprehensive reagent to finish dyeing, the osmotic pressure range of the cells in the mixed solution is 290 mmol/L-310 mmol/L.

30. A method for staining cells, characterized in that,

using the cell staining reagent of claim 13; and comprises the following steps:

step A1: diluting a sample to be dyed; adding a diluent into a sample to be dyed to prepare a sample diluent; the volume ratio range of sample and diluent is 1: 50-1: 300, respectively;

step B1: first dyeing: taking the sample diluent in the step A1, adding the first staining solution, and uniformly mixing to prepare a sample solution after primary staining; the volume ratio of the sample dilution and the first staining solution ranges from 2: 1-194: 1;

step C1: and (3) dyeing for the second time: taking the sample solution subjected to primary dyeing in the step B1, adding a second dyeing solution, and uniformly mixing to obtain a secondary dyeing sample solution; the volume ratio range of the sample liquid and the second staining liquid after the primary staining is 3: 2-49: 1.

31. a method for staining cells, characterized in that,

using the cell staining reagent of claim 13; and comprises the following steps:

step A2: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1;

step B2: preparing a comprehensive coloring agent diluent; taking the first dyeing solution diluent in the step A2, adding the second dyeing solution, and uniformly mixing to prepare a comprehensive dyeing agent diluent; the volume ratio range of the second staining solution and the first staining solution dilution is 3: 2-49: 1;

32. a method for staining cells, characterized in that,

using the cell staining reagent of claim 13;

step A3: diluting a first staining solution; adding a first dyeing solution into the diluent, and uniformly mixing to obtain a first dyeing solution diluent; the volume ratio of the dilution and first staining solution ranges from 2: 1-194: 1;

step B3: b, dyeing the sample to be dyed, and adding the first dyeing solution diluent in the step A3 into the sample to be dyed to prepare a sample solution after primary dyeing; the volume ratio range of the sample to be dyed and the sample liquid after the primary dyeing is 1: 50-1: 300, respectively;

33. the cell staining method according to any one of claims 30 to 32,

step D: spreading the dyed sample; and C, taking any one of the secondary staining sample liquid obtained in the step C1, the step C2, the step C3 or the step C3, dripping the secondary staining sample liquid into a containing carrier, uniformly spreading the secondary staining sample liquid on the containing carrier, standing for 30 seconds to 1 minute, and obtaining the staining sample for bright field observation.

34. The cell staining method according to any one of claims 30 to 32,

in the step A1, the step B1 and the step C1, in the step A2, the step B2 and the step C3, and in the step A3, the step B3 and the step C3, the mixing time is 20 seconds to 5 minutes or 30 seconds to 1 minute;

the volume of the container for staining is not less than the amount of the cell staining reagent added, while the volume of the container for staining is not more than 1 time the amount of the reagent added.

35. The cell staining method according to any one of claims 24 to 32,

also comprises the step of spreading the mixed solution after dyeing:

dripping the dyed sample liquid into a containing carrier, and uniformly spreading the dyed sample liquid on the containing carrier; after standing for 30 seconds to 1 minute, a stained sample for bright field observation was obtained.

36. A method as claimed in claim 35, wherein the holding carrier comprises a urine sediment counting plate, a cell counting plate, a viewing device made of transparent material with a cavity or a glass slide.

37. The cell staining method according to any one of claims 24 to 32,

the total dyeing time is less than 8 minutes, or the total dyeing time interval ranges from 5 minutes to 6 minutes.

38. The cell staining method according to any one of claims 24 to 32,

the sample to be dyed comprises a blood sample, a urine sample and a leucorrhea sample;

the blood sample comprises immediately collected peripheral venous blood, or EDTA (ethylene diamine tetraacetic acid) anticoagulated venous blood;

the volume of the sample to be stained is not less than 0.01 ml.

39. The cell staining method according to any one of claims 24 to 32,

the environmental temperature range for implementing the cell staining method is 15-37 ℃.