WO2022242398A1

WO2022242398A1 - Pretreatment reagent, preparation method, cell staining method and pretreatment method

Info

Publication number: WO2022242398A1
Application number: PCT/CN2022/087803
Authority: WO
Inventors: 王志平; 刘亚慧
Original assignee: 深圳安侣医学科技有限公司
Priority date: 2021-05-21
Filing date: 2022-04-20
Publication date: 2022-11-24
Also published as: CN115372107A

Abstract

Disclosed is a pretreatment reagent. The pretreatment reagent is used for the pretreatment of a cell stain in suspension, and the components thereof comprise, in percentages by volume, 0.5-5% of a stabilizer and 95-99.5% of a buffer. The components of the stabilizer comprise, in percentages by volume, 33.3-50% of a glutaraldehyde solution and 50-66.7% of a formaldehyde solution; and the pH value range of the pretreatment reagent is 6.5-8.1. On the one hand, the stabilizer enables the membrane protein on the surface of the cell membrane to be crosslinked, so that a reticular structure is formed on the surface of the cell membrane, and the cell morphology is maintained; on the other hand, the phospholipid bilayer of the cell membrane is destroyed, the permeability of the cell membrane is increased, and the efficiency of entering the cell of the subsequent dye is consistent and rapid, such that the cell staining time is shortened, the dyeing uniformity becomes high, and the reagent can be applied to various application scenarios.

Description

Pretreatment reagent and preparation method, cell staining method and pretreatment method

technical field

The present application belongs to the technical field of cell staining, and in particular relates to the cell staining technology in the state of suspension, especially to the method and reagents for pre-treatment of cell staining in the state of suspension.

Background technique

In the cell detection of biological fluids or secretions, cell staining techniques are usually used to distinguish cells of different types, shapes, and states.

In the conventional cell staining technology for bright field observation and detection, the sample to be tested is usually smeared on a transparent carrier such as a glass slide. After the dyeing is evenly completed, the main body of the dye solution and buffer solution that affect the observation is washed away, and the dyed sample is dried after waiting for the entire dyeing process to form a stained sample smear for bright field observation. The dry film staining steps in the prior art are extremely cumbersome and time-consuming, and the staining efficiency is extremely low, making it impossible to stain sample cells in large quantities. And the operation of each step requires well-trained professional operators in order to produce qualified observation samples. In addition, it takes a long time to dye the dry film and the efficiency is low, and the cost efficiency cannot meet the actual needs when testing a large number of samples.

When blood cells are stained in a liquid environment, that is, in suspension, there is no need for the complicated process of making dry films; but when staining in suspension, the presence of salt environment, organic solvents, or surfactants in the staining solution often makes blood cells Dramatic changes in shape, such as shrinkage, rupture, etc. Ultimately, after the blood cell liquid is stained in the above environment, it is impossible to output complete clinical information: such as cell quantity information, morphological information, classification information, etc. And the staining effect is easily affected by various components in the suspension, either it is difficult to stain, or it takes a long time for staining; and for different cells, it is difficult to achieve the same level of staining in a more balanced time window Effect.

名词解释：Glossary:

CAS number (CASRegistryNumber or CASNumber, CASRn, CAS#), also known as CAS registration number or CAS registration number, is the unique digital identification of a certain substance (compound, polymer material, biological sequence (Biological sequences), mixture or alloy) Number. It is constructed by the Chemical Abstracts Service (CAS) under the American Chemical Society. The agency is responsible for assigning a CAS number to each substance that appears in the literature, which avoids the trouble of having multiple names for chemical substances and makes the database search more convenient.

Purified water refers to the water that meets the purified water standard of "Chinese Pharmacopoeia (2015 Edition)". Its main parameters are: the pH value is between 4.4 and 7.6, and the off-line conductivity meter is used to detect it. Water with a conductivity not greater than 25 μS/cm (micro Siemens per centimeter) at 10 ml (milliliter).

Regis staining solution refers to the liquid dyeing solution formed by using eosin, methylene blue and azure as the main dyeing substances after certain processing technology. For example, Giemsa staining solution (Product No. BA-4017) from Zhuhai Beisuo Biotechnology Co., Ltd.

Wright's staining solution refers to the dyeing solution formed by using eosin and methylene blue as the main dyes, dissolving, mixing, and chemically acting in a certain organic solvent. For example, Wright's staining solution (article number G1040) of Beijing Suolaibao Technology Co., Ltd.

Giemsa dyeing solution refers to the dyeing solution formed after the main dyes are acid eosin and azure, which are dissolved, mixed and chemically reacted by certain organic solvents. For example, Giemsa staining solution (Product No. BA-4219) from Zhuhai Beisuo Biotechnology Co., Ltd.

New methylene blue dyeing solution refers to the dyeing solution formed by using new methylene blue as the main dye and dissolving it in a certain organic or inorganic solvent. For example, Giemsa staining solution (Product No. BA-4003) from Zhuhai Beisuo Biotechnology Co., Ltd.

Brilliant tar blue dyeing solution refers to a dyeing solution formed by dissolving brilliant cresyl blue as the main dye in a certain organic or inorganic solvent. Such as Wright's staining solution (product number G1410) of Beijing Soleibao Technology Co., Ltd.

Diff's dyeing solution refers to the dyeing solution formed by a certain process with water-soluble eosin, melanin and azure II as the main dyes and water as the main solvent. For example, Wright's staining solution (product number G1541) of Beijing Suo Laibao Technology Co., Ltd.

technical problem

The technical problem to be solved in this application is to avoid the above-mentioned shortcomings of the prior art, and propose a simple and efficient pretreatment reagent and preparation method, a cell staining method and a pretreatment method. The pre-treatment adjusts the cell state to a state suitable for suspension staining, which is very suitable for large-scale and batch sample cell staining.

technical solution

The technical scheme of the present application to solve the above-mentioned problems is a pretreatment reagent used for pretreatment of cells stained in suspension, and its components, by volume percentage, include 0.5%-5% stabilizer, 95%-99.5% buffer liquid.

The stabilizer in this solution, on the one hand, cross-links the membrane proteins on the cell membrane surface to form a network structure on the cell membrane surface to maintain the cell shape; on the other hand, it destroys the phospholipid bilayer of the cell membrane and increases the permeability of the cell membrane. The efficiency of subsequent dyes entering the cells is consistent and fast; the time for cell staining is shortened, and the uniformity of staining becomes higher.

The components of the pretreatment reagent include 1%-3% stabilizer and 97%-99% buffer in volume percentage. Preferably, the components of the pretreatment reagent include 1.75% stabilizer and 98.25% buffer in volume percentage.

The pH value range of the pretreatment reagent is 6.5-8.1; or the pH value range of the pretreatment reagent is 6.8-7.6.

The components of the stabilizer, by volume percentage, include 33.3%-50% glutaraldehyde solution, 50%-66.7% aldehyde solution; or the components of the stabilizer, by volume percentage, include 40% -50% glutaraldehyde solution, 50%-60% aldehyde solution.

Preferably, the components of the stabilizer include, by volume percentage, 42.9% glutaraldehyde solution and 57.1% formaldehyde solution.

The aldehyde substances in the aldehyde solution include any one or more of formaldehyde, acetaldehyde, propionaldehyde and paraformaldehyde.

The components of the stabilizer include, by volume percentage, 21.1%-48.3% glutaraldehyde solution and 51.7%-88.9% alcohol solution.

Alcohol substances in the alcohol solution include absolute methanol or absolute ethanol.

The glutaraldehyde solution is a glutaraldehyde solution with a mass percentage of glutaraldehyde of 50%; the aldehyde solution is an aldehyde solution with a mass percentage of aldehydes of 37%.

Preferably, the formaldehyde solution is a formaldehyde solution with a mass percent of formaldehyde of 37%.

The components of the buffer, in volume percentage, include 10%-50% phosphate buffer, 50%-90% purified water; or the components of the buffer, in volume percentage, include 20%-40 % phosphate buffer, 60%-80% purified water.

In the pretreatment reagent, the concentration of phosphate is 0.02M (mol/liter) to 0.1M (mol/liter).

Regardless of the phosphate concentration in the phosphate buffer, it is necessary to ensure that the phosphate concentration in the pretreatment reagent is from 0.02M (mol/liter) to 0.1M (mol/liter) after being prepared as a pretreatment reagent, so as to ensure the concentration of the pretreatment reagent. The pretreatment effect can not only maintain the cell shape, but also increase the permeability of the cell membrane, so that the uniformity of subsequent staining can be improved.

The components of the phosphate buffer include 5.3%-73.5% dihydrogen phosphate solution and 26.5%-94.7% hydrogen phosphate di-salt solution in volume percentage. The pH of the phosphate buffer is 6.4-8.0.

The components of the phosphate buffer include, by volume percentage, 13%-51% dihydrogen phosphate solution and 49%-87% hydrogen phosphate di-salt solution; the pH value of the phosphate buffer is 6.8.-7.6.

Preferably, the composition of the phosphate buffer solution includes 28% dihydrogen phosphate solution and 72% dihydrogen phosphate solution in volume percentage.

Preferably, the concentration of dihydrogen phosphate in the dihydrogen phosphate solution is 0.2M (mol/liter); the concentration of dihydrogen phosphate in the dihydrogen phosphate solution is 0.2M (mol/liter).

The technical solution of the present application to solve the above problems can also be a preparation method of pretreatment reagent, which is used to prepare the pretreatment reagent, including the following steps, step D: take glutaraldehyde solution and aldehyde solution with 33.3%: 66.7 % to 50%:50% volume ratio to prepare a stabilizer; Step E: Mix the stabilizer and buffer solution prepared in step D with a volume ratio of 0.5:99.5 to 5:95 to prepare a pretreatment reagent.

The preparation method of the pretreatment reagent also includes the following steps before step D: Step A: the step of preparing a 0.2M (mol/liter) dihydrogen phosphate solution; weigh the quantitative anhydrous dihydrogen phosphate, dissolve in the corresponding 0.2M (mol/liter) dihydrogen phosphate solution in 0.2M (mol/liter) dihydrogen phosphate solution; step B: the step of preparing 0.2M (mol/liter) dihydrogen phosphate solution; weigh quantitative anhydrous dihydrogen phosphate , be dissolved in the purified water of corresponding volume, and constant volume makes 0.2M (mol/liter) dihydrogen phosphate solution; The solution is mixed and prepared into a phosphate buffer solution with a pH value of 6.4-8.0; the buffer solution used in step E is the phosphate buffer solution obtained in step C; The salt concentration range is 0.02M (mol/liter) to 0.1M (mol/liter); the pH value range of the pretreatment reagent is 6.5-8.1. The above steps A and B are in no particular order.

The technical solution of the present application to solve the above problems can also be a pretreatment method, a pretreatment method, which uses the above-mentioned pretreatment reagent to pretreat the sample to be stained before cell staining; including the following steps: Step 1: the sample to be stained Mix evenly with the pretreatment reagent at a volume ratio of 1:49 to 1:399 to complete the pretreatment of the sample to be stained.

The technical solution of the present application to solve the above problems can also be a cell staining method for staining cells in a suspension; the above-mentioned pretreatment method is used to pretreat the sample to be stained before adding the staining solution.

In the cell staining method, the staining solution added to the suspension includes: Regis staining solution, Wright staining solution, Giemsa staining solution, new methylene blue staining solution, brilliant tar blue staining solution, Diff rapid staining solution any one or more of them.

Beneficial effect

Compared with the prior art, the beneficial effects of the present application are: 1. The cells in the sample to be stained, especially the blood cells in the blood sample, can be pretreated by the pretreatment reagent, and the structure of the blood cells can be preserved intact, and can be more truly Preserve the real structural information of its clinical state; 2. After the blood cells are treated with the pretreatment method, after being used with the staining solution, the state of the red blood cells and white blood cells in the blood cells after staining is relatively balanced, and can reach Uniform color degree is convenient for follow-up observation; 3. For the sample suspension to be treated after the above pretreatment reagent, the cell staining time is shortened to 1 minute to 3 minutes, and the staining efficiency is greatly improved; 4. The preparation method of the pretreatment reagent for staining It is simple, and the components are also very easy to obtain; it is a very convenient and efficient dyeing pretreatment solution; 5. After such a pretreatment process, the cell staining operation is simple, and the fool-like staining operation can be realized, making it suitable for various application scenarios . Such as immediate inspection, first aid, bedside, battlefield and other conditions that lack professionals, large equipment and complex inspection environments.

Description of drawings

Fig. 1 is a schematic flow diagram of the preparation method of the pretreatment reagent and the pretreatment method and the cell staining method of the sample to be stained with the pretreatment reagent;

Fig. 2 to Fig. 4 are respectively the micrographs obtained after tiling of the stained cell suspension obtained after the pretreatment reagent treatment in the embodiment 1 to the embodiment 3 after staining;

Figure 2 Staining effect diagrams under different cell densities, the volume ratio of the sample to the pretreatment reagent is 1:149, the number of cells in the field of view is moderate, evenly distributed, and the staining is transparent; Figure 3 The staining effect diagrams under different cell densities, the sample and The volume ratio of the pretreatment reagent is 1:49, the number of cells is large, the arrangement is tight, and the staining information of platelets is easily blocked; Figure 4 shows the staining effect under different cell densities, the volume ratio of the sample to the pretreatment reagent is 1:399, the cells The number is small, the distribution is sparse, and the staining effect is average; Figure 2, Figure 3, and Figure 4 are the renderings of staining at different densities for 4 minutes. All three ratios can obtain good cell distribution staining effects, and the effect in Figure 2 is the best .

Fig. 5 to Fig. 7 are the micrographs obtained after the stained cell suspension obtained after staining after the pretreatment reagent treatment in the embodiment 4 to the embodiment 6 respectively; Fig. 5 staining under different stabilizer concentrations Effect picture, the volume ratio of stabilizer and buffer solution is 1.75:98.25, the cells are clearly differentiated after staining; Figure 6 The staining effect picture under different concentrations of stabilizer, the volume ratio of stabilizer and buffer solution is 5:95, the cells after staining The degree of differentiation is not obvious, and the nuclei of white blood cells are lightly stained; Figure 7 shows the staining effect under different concentrations of stabilizers. The cytoplasmic discrimination is slightly poor; Figure 5, Figure 6, and Figure 7 are a set of effect diagrams of staining for 4 minutes under different stabilizer concentrations, in which the volume ratio of the three stabilizers to the buffer can obtain better staining results, Among them, in Figure 5, the red blood cell structure is intact, the white blood cell nuclear staining structure is clear, and the red blood cell is clearly distinguished from the white blood cell and platelet, and its staining is the best.

Fig. 8 to Fig. 10 are respectively the micrographs of the stained cell suspension obtained after the staining obtained after the pretreatment reagent treatment in the embodiment 7 to the embodiment 9; The lower dyeing effect diagram after the pretreatment of the treatment reagent, in embodiment 7, the phosphate concentration in the pretreatment reagent is 0.06M (mol/L), as shown in Figure 8, the cell structure is intact, and the cell differentiation degree is obvious after staining; embodiment 8 Among them, the phosphate concentration in the pretreatment reagent is 0.1M (mol/L), as shown in Figure 9, the cell structure is intact, and the degree of differentiation between the stained leukocyte nucleus and the cytoplasm is general; the phosphate concentration in the pretreatment reagent in Example 8 is 0.02M (mol/L), as shown in Figure 10, the structure of red blood cells is deformed, and the degree of differentiation between the nucleus and cytoplasm of stained white blood cells is acceptable; Figure 8, Figure 9, and Figure 10 are pretreatment reagents with different phosphate concentrations and then stained for 4 minutes The effect diagram of the effect diagram, the phosphate concentration in the pretreatment reagent is from 0.02M (mol/L) to 0.1M (mol/L), and good staining results can be obtained, in which the structure of red blood cells and white blood cells is more complete in Figure 8, and the staining of white blood cells The distinction is more obvious and its condition is optimal.

Fig. 11 to Fig. 13 are the micrographs obtained after tiling of the stained cell suspension obtained after the pretreatment reagent treatment in embodiment 10 to embodiment 12 after staining; Fig. 11 pH value of different pretreatment reagents The following staining effect diagram, when the pH value of the pretreatment reagent is 7.2, the cell structure is intact, and the differentiation of different cells is good; Figure 12 The staining effect diagram under different pH values of the pretreatment reagent, when the pH value of the pretreatment reagent is 8.0, the cell staining is too deep , the shape of the white blood cell nucleus cannot be distinguished; Figure 13 is the staining effect of different pretreatment reagent pH values. The dyeing effect diagram of 4 minutes of dyeing after pretreatment with pretreatment reagents under different pH conditions. The staining effect is better when the pH value is from 6.4 to 8.0, and the staining effect of red blood cell morphology and white blood cell in Figure 11 is better. There is not much difference between the pH of the buffer and the pH of the pretreatment reagent; the pH of the buffer dominates the pH of the pretreatment reagent.

Fig. 14 to Fig. 16 are the micrographs obtained after the stained cell suspension obtained after staining after the pretreatment reagent treatment in the embodiment 13 to the embodiment 15 respectively; Fig. 14 replaces the stabilizer with methanol formaldehyde, the volume ratio of methanol in the stabilizer is 80%; Figure 15 replaces the formaldehyde in the stabilizer with methanol, and the volume ratio of methanol in the stabilizer is 57.1%; Figure 16 replaces the formaldehyde in the stabilizer with methanol , the volume ratio of methanol in the stabilizer is 88.9%; in Figure 14, Figure 15, and Figure 16, methanol in different volume proportions is used to replace the formaldehyde in the stabilizer, and the dyeing effect of this system is better in 4 minutes, in which Figure 14 The neutrophils were uniformly stained, the nuclear outline was clear, and the red blood cells were in complete shape. Methanol includes anhydrous methanol, or methanol solutions of other concentrations that achieve the same effect as an equivalent amount of anhydrous methanol are also acceptable.

Fig. 17 to Fig. 19 are the micrographs obtained after tiling of the stained cell suspension obtained after staining after the pretreatment reagent treatment in the embodiment 16 to the embodiment 18 respectively; Fig. 17 replaces the stabilizer with ethanol formaldehyde, the volume ratio of ethanol in the stabilizer is 80%; Figure 18 replaces the formaldehyde in the stabilizer with ethanol, and the volume ratio of ethanol in the stabilizer is 57.1%; Figure 19 replaces the formaldehyde in the stabilizer with ethanol , the volume ratio of ethanol in the stabilizer is 88.9%; in Figure 17, Figure 18, and Figure 19, the formaldehyde in the stabilizer is replaced by ethanol with different volume ratios, and the dyeing effect of the system is better in 4 minutes, among which Figure 17 The neutrophils were uniformly stained, the nuclear outline was clear, and the red blood cells were in complete shape. Ethanol includes absolute ethanol, or ethanol solutions of other concentrations that achieve the same effect as an equivalent amount of absolute ethanol are also available.

The microscopic magnification of the above micrographs is 400 times; in the above micrographs, the cells are suspended in the suspension with a "three-dimensional" structure, and the positions of different cells in the suspension are different, and different cells will be in different positions. The focus plane of microscopic imaging; and the focus area of the microscope to the field of view is usually selected in the middle of the field of view, and the edge of the field of view will have virtual focus due to spherical aberration, aberration, etc., so the cell images in the edge of some pictures are slightly blurred.

Embodiments of the present invention

Embodiments of the present application will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is a schematic flowchart of the preparation method of the pretreatment reagent, the pretreatment method of the sample to be stained with the pretreatment reagent and the cell staining method.

As shown in Figure 1, the following steps are included:

Step 1: Prepare 0.2M sodium dihydrogen phosphate solution: weigh 23.99 g of anhydrous sodium dihydrogen phosphate, dissolve it in 800 ml of purified water, and dilute to 1000 ml;

Step 2: Prepare 0.2M disodium hydrogen phosphate solution; weigh 28.39 grams of anhydrous disodium hydrogen phosphate, dissolve in 800 ml of purified water, and dilute to 1000 ml;

Step 3: Prepare the optimal 0.2M phosphate buffer system; mix 0.2M sodium dihydrogen phosphate solution and 0.2M disodium hydrogen phosphate solution in a volume ratio of 28:72, and the pH value of the 0.2M phosphate buffer solution is 7.2;

Step 4: Mix the phosphate buffer solution in step: 3 with purified water in a volume ratio of 3:7 to prepare a buffer solution;

Step 5: Take 50% glutaraldehyde and 37% formaldehyde and mix them according to the volume ratio of 7.5:10 to make a stabilizer;

Step 6: Mix the phosphate buffer obtained in step 4 with the stabilizer obtained in step 5 according to the volume ratio of 1.75:98.25, and configure it as a pretreatment reagent; the phosphate concentration in the pretreatment reagent is 0.06M (mol/liter ); the pH value of the pretreatment reagent is 7.3;

Step 7: Take a fresh blood sample and mix it according to the volume ratio of sample: pretreatment reagent = 1:149, and react for 1 minute;

Step 8: Add an appropriate proportion of commercial Regis staining solution to the mixture prepared in step 7, mix well, and take pictures under a microscope.

In Example 1 to Example 3, the sample and the sample processing reagent were mixed according to the ratio of 1:49, 1:149 and 1:399 respectively, and then commercialized Regis staining solution was added to carry out the staining test; Example 2 is taken as an example, the volume ratio of sample and sample treatment solution is 1:149; the operation is as follows, take 0.005ml of fresh blood sample, add it to 0.745ml of sample treatment reagent, mix and react for 1 minute; then add 10ul commercial Thinned Rigi's staining solution, mixed evenly and then microscopically examined and photographed. Fig. 2 is the micrograph obtained after the stained cell suspension obtained after staining with the pretreatment reagent of Example 1; Fig. 3 is stained with the pretreatment reagent of Example 2 The micrograph obtained after the stained cell suspension obtained after tiling; Fig. 4 is the micrograph obtained after the stained cell suspension obtained after staining with the pretreatment reagent of Example 3 picture.

In embodiment 4 to embodiment 6, can show the influence of different stabilizing agent contents on sample pretreatment; In embodiment 4 to embodiment 6, take 50% glutaraldehyde and 37% formaldehyde as base material, prepare Stabilizer; the stabilizer and the buffer solution are formulated into sample processing reagents at volume ratios of 0.5:99.5, 5:95, and 1.75:98.25 respectively; take 0.005ml of fresh blood samples and add them to 0.495ml of sample processing reagents, Mix well and react for 1 minute; then add 10ul of commercial Regis staining solution, mix well and take pictures under microscope. Figures 5 to 7 are micrographs of the stained cell suspensions obtained after the staining was performed after the pretreatment reagents in Examples 4 to 6 were tiled.

In the pretreatment reagent preparation of embodiment 7 to embodiment 9 and pretreatment method and dyeing method, comprise the following steps:

Step 1: Mix 0.2M dihydrogen phosphate solution and 0.2M dihydrogen phosphate solution in a volume ratio of 28:72 to prepare a phosphate buffer solution with a pH value equal to 7.2 and a concentration of 0.2M (mol/liter); Step 2: Dilute 0.2M phosphate buffer solution and purified water into phosphate buffer solution according to the volume ratio of 1:9, 5:5, and 3:7 respectively; Step 3: Dilute 50% glutaraldehyde solution in the stabilizer with 37% formaldehyde solution is mixed according to the volume ratio equal to 42.9:57.1 to obtain the stabilizer; Step 4: Add the stabilizer to the above phosphate buffer respectively, the volume ratio of the phosphate buffer to the stabilizer is 98.25:1.75, before preparing Treatment reagent, at this time the phosphate concentration in the pre-treatment reagent is 0.02M, 0.1M and 0.06M respectively; pH is 7.2; Step 5: Take 0.005ml of fresh blood sample, add it to 0.495ml of sample treatment reagent, mix well and React for 1 minute; then add 10ul of commercial Regis staining solution, mix well and take pictures under microscope. Figures 8 to 10 are micrographs of the stained cell suspensions obtained after the staining was performed after the pretreatment reagents in Examples 7 to 9 were tiled.

In Examples 10 to 12, the influence of different pH environments on sample pretreatment can be seen; in Examples 10 to 12, the "0.2M phosphate buffer system" is prepared first.

In embodiment 10 to embodiment 12, comprise the following steps:

Step 1: First prepare 0.2M sodium dihydrogen phosphate solution: weigh 23.99 grams of anhydrous sodium dihydrogen phosphate, dissolve it in 800 ml of purified water, and set the volume to 1000 ml;

Step 2: Prepare a 0.2M disodium hydrogen phosphate solution; weigh 28.39 g of anhydrous disodium phosphate, dissolve it in 800 ml of purified water, and dilute to 1000 ml.

Step 3: Prepare the optimal 0.2M phosphate buffer solution; in Example 10, mix 0.2M sodium dihydrogen phosphate solution with 0.2M disodium hydrogen phosphate solution in a volume ratio of 28:72, then 0.2M phosphate buffer The pH value of the solution is 7.2; in Example 11, the 0.2M general range upper limit phosphate buffer system was prepared by mixing 0.2M sodium dihydrogen phosphate solution and 0.2M disodium hydrogen phosphate solution according to the volume ratio of 5.3:94.7. When the pH value of 0.2M phosphate buffer was 8.0; in Example 12, the 0.2M general range lower limit phosphate buffer system was prepared, and the 0.2M sodium dihydrogen phosphate solution and the 0.2M disodium hydrogen phosphate solution were according to the volume of 73.5:26.5 Ratio mixing, at this time the pH value of 0.2M phosphate buffer is 6.4;

Step 4: Mix the above three 0.2M phosphate buffers with purified water in a volume ratio of 3:7 to prepare three buffers with different pH values;

Step 5: Mix 50% glutaraldehyde solution and 37% formaldehyde solution in the stabilizer according to the volume ratio equal to 42.9:57.1 to obtain the stabilizer;

Step 6: Add the stabilizer to the above-mentioned phosphate buffer with pH values of 7.2, 8.0, and 6.4 respectively, the volume ratio of the phosphate buffer to the stabilizer is 98.25:1.75, and prepare the pretreatment reagents of Examples 10 to 12 respectively ; Now the phosphate concentration in the pretreatment reagent is 0.06M, and the pH value of the pretreatment reagent is 7.3, 8.1, 6.5 respectively;

Step 7: Take another 0.005ml fresh blood sample, add it to 0.495ml sample processing reagent, mix well and react for 1 minute;

Step 8: Add 10ul of commercial Regis staining solution, mix well and take pictures under microscope. Figures 11 to 13 are micrographs of the stained cell suspensions obtained after being stained after being treated with the pretreatment reagents in Examples 10 to 12, respectively.

In embodiment 13 to embodiment 15, in the pretreatment reagent preparation and pretreatment method and dyeing method, comprise the following steps:

Step 1: Mix 0.2M dihydrogen phosphate solution and 0.2M dihydrogen phosphate solution in a volume ratio of 28:72 to prepare a phosphate buffer solution with a pH value equal to 7.2 and a concentration of 0.2M (mol/liter);

Step 2: Dilute 0.2M phosphate buffer solution and purified water into phosphate buffer solution at a volume ratio of 3:7;

Step 3: Mix 50% glutaraldehyde solution and anhydrous methanol solution in the stabilizer according to volume ratios equal to 20%: 80%, 42.9%: 57.1%, 11.1%: 88.9%, respectively, to obtain the stabilizer;

Step 4: Add the stabilizer to the above-mentioned phosphate buffer respectively, the volume ratio of the phosphate buffer to the stabilizer is 98.25:1.75, and prepare the pretreatment reagent; at this time, the phosphate concentration in the pretreatment reagent is 0.02M, 0.1M M and 0.06M, the pH value of the pretreatment reagent is 7.2; take 0.005ml fresh blood sample, add it to 0.495ml sample treatment reagent, mix and react for 1 minute; then add 10ul of commercial Regis staining solution, mix After uniformity, take pictures under the microscope. Figures 14 to 16 are micrographs of the stained cell suspensions obtained after the staining was performed after the pretreatment reagents in Examples 13 to 15 were tiled.

In embodiment 16 to embodiment 18 in the pretreatment reagent preparation and pretreatment method and dyeing method, comprise the following steps:

Step 3: Mix 50% glutaraldehyde solution and absolute ethanol solution in the stabilizer according to volume ratios equal to 20%: 80%, 42.9%: 57.1%, 11.1%: 88.9%, respectively, to obtain the stabilizer;

Step 4: Add the stabilizer to the above-mentioned phosphate buffer respectively, the volume ratio of the phosphate buffer to the stabilizer is 98.25:1.75, and it is prepared into the pretreatment reagents of Examples 16 to 18. At this time, the phosphate concentration in the pretreatment reagent Both are 0.06M, and the pH value is 7.3;

Step 5: Take 0.005ml of fresh blood sample, add it to 0.495ml of sample processing reagent, mix well and react for 1 minute; then add 10ul of commercial Regis staining solution, mix well and take pictures under microscope. Figures 17 to 19 are micrographs of the stained cell suspensions obtained after the staining was performed after the pretreatment reagents in Examples 16 to 18 were tiled.

Among the pretreatment reagents and pretreatment methods, cell staining reagents and methods in the present application, the technical effects include multiple aspects. A fresh sample of body fluid or secretion in which the cells are biologically active. The pretreatment of the present application can cross-link the membrane protein on the surface of the cell membrane to form a network structure on the surface of the cell membrane to maintain the cell shape, while destroying the phospholipid bilayer of the cell membrane and increasing the permeability of the cell membrane; After treatment, on the one hand, it can maintain a good cell shape, and at the same time, by maintaining the cell shape, it also maintains the activity of the internal substances in the cell, so that the internal environment inside the cell is closer to the original physiological environment of the cell, and the time for maintaining the activity of the organelles inside the cell will be longer. longer. After the staining solution enters the cell, it can exert the activity of the internal organelles of the cell and achieve the purpose of rapidly staining the cell.

The cell staining reagent and staining method designed in the present application can complete cell staining in body fluid or secretion in liquid suspension. Its applicable samples include various biological fluids such as blood, secretions such as urine or leucorrhea, etc.

In the cell staining method in this application, the cells in the sample to be stained complete the staining process in a liquid suspension, and the environment used for the staining solution and diluent is relatively close to the biological physiological state, so in the staining process, the cell activity On the one hand, it can use the Brownian motion of the cells and the dye molecules in the solution and the electrostatic force to carry out the dyeing reaction, and the dyeing speed is faster and the efficiency is higher; on the other hand, due to such a dyeing environment, The cell viability is kept relatively well, and fast and balanced cell staining can be achieved through the pre-treated samples to be stained. It is especially suitable for the pre-treatment of live cell staining, which can greatly improve the efficiency of cell staining.

In the method of this application, the ratio between the sample staining reagent and the sample is set reasonably, and the living cells in the sample can be stained. The operation is simple, the staining is fast, the staining effect is better, and the staining result is more conducive to the shape of the cells. The stained solution can be directly applied to the analysis and detection of cell morphology under bright field, and can further carry out cell classification and cell classification counting according to the analysis of the stained cell morphology and graphic characteristics.

In the field of medical applications, it has not been seen that cells are in a solution state to carry out live dyeing of blood samples in vitro, that is, using compound dyes with chromophores to directly stain blood cells, and based on blood cells under a bright field microscope The cell characteristics presented in the staining results were analyzed for cell classification. The body fluids or secretions stained by the method of the present application show cell characteristics including the size, color, shape, shape and color of the nucleus, the color of the cytoplasm, the color and the number of particles in the cytoplasm. The stained sample in this application is suitable for morphological analysis of cells, based on the analysis of cell characteristics such as cell morphology and coloring degree of staining, cell classification, identification and counting can be performed.

Compared with the staining methods and reagents in the prior art, the main feature of the present application is that the state of the sample cells is in a physiological or close to physiological state when staining in the present application. Compared with the dyeing methods and reagents in the prior art, the main feature of this application is that the reaction environment during dyeing is completely different from the operation, and this application does not involve the process of drying and cleaning; in this application, the cells in the sample Always in a liquid solution environment, after pretreatment with the sample of pretreatment reagents, various target cells to be stained have more balanced staining characteristics, and the staining reaction is carried out, the staining efficiency is higher, and the staining efficiency of different cells is more balanced.

The above reagents and methods can perform efficient pretreatment and efficient staining of the active cells in the sample, have low requirements on the operator, uniform staining, and short time-consuming, and can realize a foolish staining process; avoiding the need for cells in the prior art The dyeing operation is cumbersome, the professional requirements are high, the time is long, the dyeing is easy to be uneven, and the dyeing material cost is high. The pretreatment and dyeing operations are simple, and can realize fool-like dyeing operations, making it suitable for various application scenarios. Scenarios such as instant inspection are also applicable to various application scenarios that lack large-scale professional equipment, professionals, and complex inspection environments, such as first aid, bedside, and battlefield.

The above is only an embodiment of the application, and does not limit the patent scope of the application. Any equivalent structure or equivalent process conversion made by using the description of the invention and the contents of the accompanying drawings, or directly or indirectly used in other related technical fields , are all included in the patent protection scope of the present application in the same way.

Claims

A pretreatment reagent, characterized in that,

For pretreatment of samples to be stained before cells are stained in suspension,

Its components, by volume percentage, include 0.5%-5% stabilizer and 95%-99.5% buffer.
The pretreatment reagent according to claim 1, characterized in that,

Its components, by volume percentage, include 1%-3% stabilizer and 97%-99% buffer.
The pretreatment reagent according to claim 1 or 2, characterized in that,

The pH range of the pretreatment reagent is 6.5-8.1;

Or the pH range of the pretreatment reagent is 6.8-7.6.
The pretreatment reagent according to claim 1 or 2, characterized in that,

The components of the stabilizer, in volume percentage, include 33.3%-50% glutaraldehyde solution, 50%-66.7% aldehyde solution;

Or the components of the stabilizer, by volume percentage, include 40%-50% glutaraldehyde solution and 50%-60% aldehyde solution.
The pretreatment reagent according to claim 4, characterized in that,

The aldehyde substances in the aldehyde solution include any one or more of formaldehyde, acetaldehyde, propionaldehyde and paraformaldehyde.
The pretreatment reagent according to claim 1 or 2, characterized in that,

The components of the stabilizer include, by volume percentage, 21.1%-48.3% glutaraldehyde solution and 51.7%-88.9% alcohol solution.
The pretreatment reagent according to claim 6, characterized in that,

Alcohol substances in the alcohol solution include absolute methanol or absolute ethanol.
The pretreatment reagent according to claim 4, characterized in that,

Described glutaraldehyde solution is the glutaraldehyde solution that the mass percent of glutaraldehyde is 50%;

The aldehyde solution is an aldehyde solution with a mass percentage of aldehyde substances of 37%.
The pretreatment reagent according to claim 1 or 2, characterized in that,

The components of the buffer include, by volume percentage, 10%-50% phosphate buffer, 50%-90% purified water;

Or the components of the buffer, by volume percentage, include 20%-40% phosphate buffer, 60%-80% purified water.
The pretreatment reagent according to claim 9, characterized in that,

In the pretreatment reagent, the concentration of phosphate is 0.02M (mol/liter) to 0.1M (mol/liter).
The pretreatment reagent according to claim 9, characterized in that,

The components of the phosphate buffer include 5.3%-73.5% dihydrogen phosphate solution and 26.5%-94.7% hydrogen phosphate di-salt solution in volume percentage.
The pretreatment reagent according to claim 11, characterized in that,

The components of the phosphate buffer include, by volume percentage, 13%-51% dihydrogen phosphate solution and 49%-87% hydrogen phosphate di-salt solution.
A method for preparing a pretreatment reagent, characterized in that,

For preparing the pretreatment reagent described in claim 4, comprising the following steps,

Step D: Mix glutaraldehyde solution and aldehyde solution at a volume ratio of 33.3%:66.7% to 50%:50% to prepare a stabilizer;

Step E: Mix the stabilizer and buffer prepared in step D with a volume ratio of 0.5:99.5 to 5:95 to prepare a pretreatment reagent.
The preparation method of pretreatment reagent according to claim 13, characterized in that,

The following steps are also included before step D:

Step A: the step of preparing 0.2M (mol/liter) dihydrogen phosphate solution; weigh quantitative anhydrous dihydrogen phosphate, dissolve it in a corresponding volume of purified water, and prepare 0.2M (mol/liter) dihydrogen phosphate hydrogen salt solution;

Step B: the step of preparing 0.2M (mol/liter) hydrogen phosphate di-salt solution; weigh quantitative anhydrous hydrogen phosphate di-salt, dissolve it in a corresponding volume of purified water, and prepare 0.2M (mol/liter) hydrogen phosphate di-salt solution;

Step C: mixing the dihydrogen phosphate solution prepared in step A and the dihydrogen phosphate di-salt solution prepared in step B to prepare a phosphate buffer solution with a pH value of 6.4-8.0;

The buffer used in step E is the phosphate buffer that step C makes;

The dosage ratio of the phosphate buffer is such that in the prepared pretreatment reagent, the phosphate concentration is 0.02M (mol/liter) to 0.1M (mol/liter); the pH value range of the pretreatment reagent is 6.5-8.1.
A pretreatment method, characterized in that,

Used the pretreatment reagent described in any one in claim 1 to 12 to carry out the pretreatment before cell staining of the sample to be stained; comprise the following steps:

Step 1: Mix the sample to be stained and the pretreatment reagent evenly at a volume ratio of 1:49 to 1:399 to complete the pretreatment of the sample to be stained.
A cell staining method, characterized in that,

For staining cells in suspension;

The pretreatment method in claim 14 is used to pretreat the sample to be dyed before adding the staining solution.
The cell staining method according to claim 16, characterized in that,

The staining solution added to the suspension includes any one or more of Rigi’s staining solution, Wright’s staining solution, Giemsa staining solution, new methylene blue staining solution, brilliant tar blue staining solution, and Diff’s rapid staining solution kind.