CN112213172A

CN112213172A - Stable vaginal secretion visible component staining solution and preparation method thereof

Info

Publication number: CN112213172A
Application number: CN202010974246.4A
Authority: CN
Inventors: 朱子是; 孙德亚; 林月; 赵亚荣; 王嘉星; 高威; 何浩会
Original assignee: Dirui Medical Technology Co Ltd
Current assignee: Dirui Medical Technology Co Ltd; Changchun Dirui Industrial Co Ltd
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2021-01-12

Abstract

The invention relates to a stable staining solution for visible components of vaginal secretion and a preparation method thereof, belonging to the technical field of staining solutions. The problems of complex dyeing steps, complex operation, low dyeing speed, poor using effect, high cost and small daily film reading amount in the prior art are solved. The stable vaginal secretion visible component staining solution comprises a staining solution A and a staining solution B; the dyeing liquid A comprises an organic solvent and a dyeing agent, wherein the dyeing agent is a red system dyeing agent, a green system dyeing agent, a purple system dyeing agent or a blue system dyeing agent; the dyeing liquid B comprises a buffer solution, a preservative, a dyeing assistant, an osmotic pressure regulator and a surfactant. The staining solution has excellent cell detection morphology, high accuracy and good stability, and the effective period can reach 12 months.

Description

Stable vaginal secretion visible component staining solution and preparation method thereof

Technical Field

The invention belongs to the technical field of staining solutions, and particularly relates to a stable staining solution for visible components of vaginal secretion and a preparation method thereof.

Background

Vaginal secretion, the most common method in medical clinical examination, is optical microscopy, before examination, the collected original sample is filtered, diluted and mixed uniformly to meet the requirement of microscopic examination, and then the sample is prepared into a slide specimen suitable for microscopic examination, and the specimen is divided into a dry sheet and a wet sheet. The wet sheet preparation method mainly comprises a counting cell method and a cover glass sheet method. The counting cell method is that sample liquid (dyed or not dyed) is added into an optical counting cell, then microorganisms (cells) are fixed at an observable position by methods such as natural sedimentation or artificial sedimentation, and then the counting cell is sent to a microscope for observation. The cover glass method is that the sample liquid is dripped on the glass slide, then the cover glass is covered to form a liquid thin layer with a certain thickness, and then the counting cell is sent to a microscope for observation. By checking cells, microorganisms or other tangible components in a target, the number and the state of the tangible components are often used as judgment conditions for judging whether the female vagina is healthy, and important reference information is provided for medical diagnosis. However, no cell staining component is added under microscopic examination, so that the microscopic examination level requirement of a clinician is high, the misjudgment rate is high, and the visual fatigue feeling of the clinician due to long-term work is increased, so that the detection accuracy and the detection effect are influenced.

The existing staining method is Papanicolaou and HE, belongs to a cytopathology staining method, and has the advantages of long fixing time and long staining time, but the staining steps are complicated, the waiting time is long, the report outputting speed is slow, the daily film reading amount is small, the cost is high, the operation is complex, and the method is labor-consuming, time-consuming and expensive.

The Jimsa staining method is also an emerging staining method, is mainly applied to cytological examination of conjunctiva of eyes and cytological diagnosis of clinical malignant tumors, has the advantages of high speed, only needs 3 minutes, has good staining effect on cell nucleus, and has the defect of poor staining effect on cytoplasm, cell membranes and neutral particles in the cell cytoplasm and cell membranes.

The improved Ruhry-Jimsang mixed staining method is an improvement on a pure Jimsang staining method, and is mainly used for blood smear examination, the staining method comprises the steps of firstly selecting a dyeing solution of Ruhry for 25 minutes, removing the dyeing solution of Ruhry, then dyeing for 4 minutes by the dyeing solution of Jimsang, washing by purified water, and placing in the air for self-drying, and the method overcomes the defect that the staining method of Jimsang has poor staining effects on cytoplasm and cell membranes, but medical staff are complicated to operate, and the staining speed of the method is slow, and needs more than 30 minutes.

The leucorrhea microscopic examination is one of the current clinical routine laboratory examination items, and various biological cell conditions in the leucorrhea reflect the pathological changes of the female vagina. At present, most of small hospitals in China still use a microscope for detection in leucorrhea detection and then carry out manual treatment, but the manual detection is greatly influenced by human factors, so that the method is only suitable for detection in a small range. For the detection with larger workload, the detection personnel are easy to fatigue after working under the microscope for a long time, thereby influencing the judgment of medical personnel on the state of an illness. Meanwhile, the doctor has a large subjective influence on the analysis of the cell pattern, and the doctor can easily judge the disease condition by the own experience. Therefore, in the detection of the leucorrhea, the introduction of automatic detection equipment to replace manual detection is necessary, so that the detection accuracy and the working efficiency can be improved, and the interference of subjective factors can be eliminated by quantitative objectivity to ensure the detection quality. With the progress of digital image processing technology, many fields begin to use digital image processing, and the medical field is no exception, and the digital image processing brings substantial changes to medical diagnosis, thereby promoting the revolution of medical diagnosis. The overall design of the GMD-S600 full-automatic gynecological secretion analyzer independently developed by diry meets the microscopic examination requirements of medical clinic, the morphological characteristics (cell morphology, cell size, nuclear quantity, nuclear morphology, nuclear size, nuclear plasma volume ratio, cell boundary, refraction characteristic and the like) of various visible components in the white band are summarized and described respectively, and related cell maps are consulted to provide different cell morphological shape surface schematic diagrams and different cell staining effect pictures, so that related non-professionals can conveniently know, familiarize and know various visible components which can be detected in the white band, and software designers can conveniently design image identification parameters according to different morphological visible component types and different characteristics of cells with the same type and different growth cycles, so that a white band analyzer can more accurately and detailedly identify visible components in the white band detection, and the detection rate and accuracy are improved. The components of the leucorrhea include leukocytes, epithelial cells, bacilli and cocci, and are classified accurately according to morphological features of the respective cells (see table 1).

TABLE 1 cellular diagnostic basis and clinical significance

The automatic gynecological secretion analyzer solves the existing problems of complicated operation, environmental pollution, great randomness and the like of manual leucorrhea detection, enables the operation to be automatic and standard, realizes rail-type sample introduction, enables samples to be detected at any time, more importantly solves the great problem of biological safety, improves the working environment, has very important social value and practical significance for the research of the leucorrhea biological cell medical microscopic image automatic identification technology, and the dyeing liquid is a necessary reagent for ensuring the analyzer to detect visible components and influences the completeness, accuracy and reliability of detection.

Disclosure of Invention

The invention aims to solve the problems of complicated dyeing steps, complex operation, low dyeing speed, poor using effect, high cost and small daily film reading amount in the prior art, and provides a stable dyeing solution for visible components of vaginal secretion and a preparation method thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows.

The stable vaginal secretion visible component staining solution comprises a staining solution A and a staining solution B;

the dyeing liquid A comprises an organic solvent and a dyeing agent, wherein the dyeing agent is a red system dyeing agent, a green system dyeing agent, a purple system dyeing agent or a blue system dyeing agent;

the dyeing liquid B comprises a buffer solution, a preservative, a dyeing assistant, an osmotic pressure regulator and a surfactant.

Preferably, the organic solvent is one or a mixture of more of methanol, ethanol, ethylene glycol, propanol, glycerol, butanol, pentanol, acetone, acetonitrile, N-dimethylformamide and dimethyl sulfoxide.

Preferably, the red system staining agent is one or a mixture of more of neutral red, eosin G, eosin Y, thiazine dyes, Sudan III, red acetate, tangerine G and hematoxylin;

the green system coloring agent is one or a mixture of more of fast green and methyl green;

the purple system coloring agent is one or a mixture of gentian violet and crystal violet;

the blue system coloring agent is one or a mixture of toluidine blue, methylene blue, azure II, Coomassie brilliant blue and methylene blue;

in the dyeing liquid A, the concentration of the dyeing agent is 0.5-2.0 wt%.

Preferably, the buffer is one or a mixture of several of HEPES buffer, PBS buffer, MOPSO buffer, BTIS buffer and Tris buffer, the pH value of the buffer is 5.0-9.0, and the concentration of the buffer is 10mM-100 mM.

Preferably, the preservative is one or a mixture of more of sodium azide, thimerosal, gentamicin, ProClin300, 2-methyl-4-isothiazoline-3-ketone, sodium pyrithione and sodium dehydroacetate, and the concentration of the preservative is 0.1-0.75 wt%.

Preferably, the dyeing assistant is one or a mixture of more of calcium acetate, sodium acetate, amine acetate, potassium acetate and magnesium acetate, and the concentration of the dyeing assistant is 10mM-100 mM.

Preferably, the osmotic pressure regulator is sodium chloride or potassium chloride, and the concentration of the osmotic pressure regulator is 0.5 wt% to 1.5 wt%.

Preferably, the surfactant is one or a mixture of more of polyoxyethylene lauryl ether, triton X-100, BRIJ35, BRIJ98, EMULGEN24B, span 60, Tween 20, Tween 40, poloxamer, triton X-100 and octylphenol polyoxyethylene ether, and the concentration of the surfactant is 0.01 wt% -1.00 wt%.

Preferably, the pH values of the stain A and the stain B are respectively and independently 6.0-7.0(25 +/-1) DEG C; the electric conductivities are respectively and independently 7.00 +/-0.20 mS/cm (25 +/-1) DEG C; the osmotic pressures were 185. + -.10 mmol/L (mOsm/kg), respectively.

The invention also provides a preparation method of the stable vaginal secretion visible component staining solution, which comprises the following steps:

weighing the components in the staining solution B according to the composition, uniformly stirring, adjusting the pH to 6.50 +/-0.50 (25 +/-1 ℃) by using acetic acid or 1M sodium hydroxide, adjusting osmotic pressure and conductivity, and filtering to obtain the staining solution B;

and weighing the components in the staining solution A according to the composition, uniformly stirring, and filtering to obtain the staining solution A.

Compared with the prior art, the invention has the beneficial effects that:

1. the stable staining solution for visible components of vaginal secretion has the advantages of excellent cell detection form, high accuracy and good stability, and the validity period can reach 12 months.

2. The stable vaginal secretion visible component staining solution provided by the invention has the advantages that the dye and the concentration of the dye can carry out specific staining on cytoplasm and cell nucleus, and specific differentiation is carried out; the dyeing assistant can stably dye various cells without generating a decoloration phenomenon; the pH value of the staining solution and the concentration of the buffer solution are selected according to the difference of the isoelectric points of membrane proteins of different cells and the difference of the attraction capacity of positive and negative charges, so that the adsorption capacity of various cells on the dye is optimal, the higher the concentration of the buffer solution is, the higher the ionic strength is, the stronger the interaction between ions is, and the ions in the dye can be fully combined with cell components; the surfactant has the functions of wetting, dispersing, emulsifying, solubilizing, moisturizing, permeating, antisepsis and the like, is active on the surface and the interface, and has extremely high capacity and efficiency of reducing surface and interface tension.

3. The stable visible component staining solution for the vaginal secretion is combined with a GMD-S600 full-automatic gynecological secretion analysis system, so that the definition of an interface background is improved, a background after staining is clear, the cell permeability is good, and the color of various cells is obviously distinguished.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a process flow chart of the preparation method of the coloring liquid for visible components of the stable vaginal secretion.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.

The invention idea of the invention is as follows:

firstly, the analysis of the tested object is the key core of the development of reagent products and is a prerequisite condition of the formulation design, and the molecular structure and the physicochemical property of different tested objects have obvious difference. The detection principle, methodology and technical route of the reagent are different according to different detected objects. Only if the molecular structure and physicochemical properties of the object to be detected are fully known and grasped can the detection method be correctly selected and the technical route be designed. Only if the molecular structure and the physicochemical property of the object to be measured are fully known, the stability of the molecule can be correctly evaluated, and a theoretical basis is provided for the subsequent formula design of the staining solution.

Secondly, from the perspective of the properties, the content, the physicochemical indexes and the like of the visible components in the secretion, various substances sold in the market are fully researched and analyzed, morphological characteristics (cell morphology, cell size, nucleus quantity, nucleus morphology, nucleus size, nuclear plasma volume ratio, cell boundary, refractive characteristics and the like) of the visible components in the leucorrhea are respectively summarized and described, and related cell maps are consulted to provide different cell morphological shape maps and different cell staining effect pictures, so that related laymen can conveniently know, familiarize and recognize various possible detected visible components in the leucorrhea, software designers can conveniently design image identification parameters according to different morphological visible component types and different characteristics of cells in the same type and different growth cycles, for example, staining solution is a main evaluation index mainly for staining of cell nucleuses and cytoplasm in the visible components, taking neutral red dyeing as an example, the dyeing principle is as follows: the main component of the nucleus is deoxyribonucleic acid, and the phosphate group of the deoxyribonucleic acid faces outwards, is negatively charged and is easily combined with positively charged neutral red, so that the nucleus is dyed red. The color of the nucleus is proportional to the density of DNA. The staining effect is that cytoplasm is not colored or is colored in light pink or light red, and nucleus is colored in dark red, so that visible components in the leucorrhea detection can be identified more accurately and in detail by a leucorrhea analyzer, and the detection rate and accuracy are improved.

Moreover, after the dyeing liquid components meeting the standard are screened out, the formula is integrally adjusted, the good uniformity, accuracy and stability of the reagent in the testing process are ensured, repeated experimental verification is carried out, the cell imaging is ensured to be clear and accurate, the mutual adhesion among cells is reduced, the nucleus quality is clear and identifiable on a secretion analyzer in a systematic evaluation mode, and finally the dyeing liquid formula with excellent cell shape, high accuracy and good stability is screened out.

wherein the dyeing liquid A comprises an organic solvent and a dyeing agent, and the dyeing agent is a red system dyeing agent, a green system dyeing agent, a purple system dyeing agent or a blue system dyeing agent; the appearance is reddish brown liquid without sediment, particles or floccules;

the dyeing liquid B comprises a buffer solution, a preservative, a dyeing assistant, an osmotic pressure regulator and a surfactant; the appearance is colorless transparent liquid without sediment, particles or floccules.

In the technical scheme, the organic solvent is preferably one or a mixture of more of methanol, ethanol, glycol, propanol, glycerol, butanol, pentanol, acetone, acetonitrile, N-dimethylformamide and dimethyl sulfoxide; more preferably a mixture of ethanol and ethylene glycol, a mixture of propanol and glycerol, a mixed solvent of ethylene glycol and methanol; most preferred is a mixture of ethylene glycol and methanol.

In the above technical scheme, the red system staining agent is preferably one or a mixture of more of neutral red, eosin G, eosin Y, thiazine dyes, Sudan III, red poplar, red orange G and hematoxylin; more preferably one or a mixture of more of neutral red, eosin G, eosin Y and Sudan III; particularly preferably neutral red, eosin G or eosin Y; most preferably neutral red.

In the technical scheme, the blue system coloring agent is preferably one or a mixture of several of toluidine blue, methylene blue, azure II, Coomassie brilliant blue and methylene blue; more preferably toluidine blue, azure II or methylene blue; most preferred is azure II.

In the above technical scheme, the violet system coloring agent is preferably one or a mixture of gentian violet and crystal violet.

In the above technical solution, the green system coloring agent is preferably one or a mixture of more of fast green and methyl green.

In the above technical solution, the concentration of the dyeing agent in the dyeing liquid a is preferably 0.5 to 2.0 wt%, and more preferably 1.5 wt%.

In the above technical scheme, the buffer solution is preferably one or a mixture of several of a HEPES buffer solution, a PBS buffer solution, a MOPSO buffer solution, a BTIS buffer solution, and a Tris buffer solution, and more preferably is a BTIS buffer solution; the pH of the buffer is preferably 5.0 to 9.0, more preferably 6.5; the concentration of the buffer is preferably 10mM-100mM, more preferably 50 mM.

In the technical scheme, the preservative is preferably one or a mixture of more of sodium azide, thimerosal, gentamicin, ProClin300, 2-methyl-4-isothiazoline-3-one, sodium pyrithione and sodium dehydroacetate, and more preferably 2-methyl-4-isothiazoline-3-one; the concentration of the preservative is preferably 0.1 wt% to 0.75 wt%, more preferably 0.2 wt%.

In the above technical scheme, the dyeing auxiliary is preferably one or a mixture of more of calcium acetate, sodium acetate, ammonium acetate, potassium acetate and magnesium acetate, and more preferably calcium acetate; the concentration of the dyeing auxiliary is preferably 10mM-100mM, more preferably 60 mM.

In the above technical scheme, the osmotic pressure regulator is preferably sodium chloride or potassium chloride; the concentration of the osmotic pressure regulator is 0.5 wt% to 1.5 wt%, more preferably 0.7 wt% to 0.9 wt%.

In the technical scheme, the surfactant is preferably one or a mixture of more of polyoxyethylene lauryl ether, triton X-100, BRIJ35, BRIJ98, EMULGEN24B, span 60, Tween 20, Tween 40, poloxamer, triton X-100 and octylphenol polyoxyethylene ether, and more preferably the surfactant is triton X-100; the concentration of the surfactant is preferably 0.01 wt% to 1.00 wt%, more preferably 0.05 wt%.

In the above technical solution, preferably, the pH values of the stain a and the stain B are respectively and independently 6.0-7.0(25 ± 1) ° c; the electric conductivities are respectively and independently 7.00 +/-0.20 mS/cm (25 +/-1) DEG C; the osmotic pressures were 185. + -.10 mmol/L (mOsm/kg), respectively.

weighing the components in the staining solution B according to the composition, uniformly stirring, and adjusting the pH to 6.50 +/-0.50 (25 +/-1 ℃) by using acetic acid or 1M sodium hydroxide; regulating the regulation requirement range of osmotic pressure and conductivity, and filtering to obtain a dyeing solution B;

In the above technical scheme, preferably, each component in the staining solution B is sequentially added into a container to be dissolved and stirred, one component is dissolved and then the other component is added, and after all the components are dissolved, stirring is performed for 20 minutes. The stirring is carried out by a magnetic stirrer or an overhead stirrer.

In the above technical scheme, preferably, each component in the staining solution a is heated and stirred for 2 hours at 60 ℃, and a magnetic stirrer or an overhead stirrer is adopted for stirring. .

In the above technical means, the filtration pore size is preferably 0.1 μm.

In the above technical solution, preferably, the method further comprises dispensing the staining solution a and the staining solution B.

The invention discloses a method for operating and using a stable vaginal secretion visible component staining solution, which comprises the following steps: placing the staining solution A and the staining solution B into a centrifuge tube according to the volume ratio of 3:7, shaking up, and recording as staining solution AB for later use; meanwhile, treating the secretion clinical sample by using a cell preservation solution for later use; the staining solution AB and the clinical sample are mixed evenly according to the proportion of 1:4, 20 mul is put on a glass slide, a cover glass is covered, and the cell staining condition is observed under a microscope under 40 times of ocular lens.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The stable vaginal secretion visible component staining solution comprises a staining solution A and a staining solution B, and the formula is as follows:

the preparation method of the stable vaginal secretion visible component staining solution comprises the following steps:

preparation before weighing:

selecting instruments and glassware with proper measurement precision, such as electronic balance, electronic scale, graduated cylinder, stainless steel spoon, and confirming the instruments and utensils before weighing.

Weighing and dissolving: the dyeing liquid B is prepared by weighing the chemical drugs in sequence according to the preparation process, adding the next chemical drug after each chemical drug is completely dissolved, stirring and dissolving by using a magnetic stirrer or an overhead stirrer, continuously stirring for 20 minutes after all the chemical drugs are dissolved, and adjusting the pH to be 6.50 +/-0.50 (25 +/-1 ℃) by using acetic acid or 1M sodium hydroxide; regulating osmotic pressure and conductivity.

The dyeing solution A is prepared by weighing chemical drugs and solvents in turn according to a preparation process, wherein a used container is strictly anhydrous, heating and stirring are carried out for 2 hours at 60 ℃ while mechanical stirring is carried out, and a thermometer is inserted below the liquid level to monitor the temperature of the reagent.

Filtering: the polyether sulfone filter element with the diameter of 0.1 mu m is well installed, the filter element is installed on a base of the sleeve, the filter element rotates rightwards by 45 degrees to 90 degrees, the rubber pad, the sleeve and the cock are sequentially installed, the buckle is screwed down, and no liquid leakage phenomenon is ensured. Installing a peristaltic pump, wherein the starting speed is 100 and 200 revolutions per minute;

fourthly, subpackaging: and (3) subpackaging the dye solution B into 100mL of reagent by using a peristaltic pump, subpackaging 100mL of reagent in each bottle, setting the parameters of the peristaltic pump as 100-.

And (3) subpackaging the dye solution A into 100ml brown bottles by using a peristaltic pump, wherein each bottle is 100ml, the setting parameter of the peristaltic pump is 100-.

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

c. filling out a field clearing record, a field clearing person, a date and an instrument use record;

the equipment is detailed by a high-density polyethylene bottle, a 10 ten thousand grade electronic balance, a PH meter, an osmometer, a conductivity meter, a 1000mL measuring cylinder, a 0.1 mu m polyethersulfone filter element, a special filter and a peristaltic pump.

Example 2

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 3

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 4

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 5

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 6

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 7

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 8

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 9

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

Example 10

preparation before weighing:

Fifthly, cleaning the field

a. All chemical reagents are returned; counting the medicines;

b. conveying some containers to a cleaning room for cleaning;

The performance of the stable vaginal discharge body-forming staining solutions of example 1, example 3, example 6 and example 9 was evaluated.

(iii) evaluation of appearance

The detection method comprises the following steps: randomly extracting 5 bottles of staining solution A to check appearance performance indexes by normal vision, wherein the staining solution A meets the requirements: dyeing A is a red brown liquid without sediment, particles or floccules;

randomly extracting 5 bottles of staining solution B to check appearance performance indexes by normal vision, wherein the staining solution B meets the requirements: the dyeing B is colorless transparent liquid without sediment, particles or floccules.

The stable vaginal secretion of the example 1, the example 3, the example 6 and the example 9 is qualified by the visual inspection result of the visible component staining solution.

② evaluation of accuracy

The detection method comprises the following steps: using the staining solution of example 1, 5 random samples of the secretion were tested to stain each type of cell in vaginal secretion without staining cytoplasm or with light pink or red color and staining nuclei with deep red color. The results are shown in Table 2.

The operation method comprises the following steps: placing the staining solution A and the staining solution B in a 1mL centrifuge tube according to the ratio of 3:7 (300. mu.l: 700. mu.l), shaking up, and recording as a staining solution AB for later use; meanwhile, treating the secretion clinical sample by using a cell preservation solution for later use; the staining solution AB and the clinical sample are mixed evenly according to the proportion of 1:4, 20 mul is put on a glass slide, a cover glass is covered, and the cell staining condition is observed under a microscope under 40 times of ocular lens.

Table 2 dyeing liquor accuracy evaluation data of example 1

The detection method comprises the following steps: using the staining solution of example 3, 5 random samples of the secretion were tested to stain each type of cell in vaginal secretion without staining cytoplasm or with light pink or red color and staining nuclei with deep red color. The results are shown in Table 3.

The operation method comprises the following steps: and (3) mixing the staining solution A and the staining solution B according to the weight ratio of 3:7 (300. mu.l: 700. mu.l) in a 1mL centrifuge tube, shaking up, and recording as a staining solution AB for later use; meanwhile, treating the secretion clinical sample by using a cell preservation solution for later use; the staining solution AB and the clinical sample are mixed evenly according to the proportion of 1:4, 20 mul is put on a glass slide, a cover glass is covered, and the cell staining condition is observed under a microscope under 40 times of ocular lens.

Table 3 dyeing liquor accuracy evaluation data of example 3

The detection method comprises the following steps: using the staining solution of example 6, 5 random samples of the secretion were tested to stain each type of cell in vaginal secretion without staining cytoplasm or with light or sky blue color and with staining nuclei with dark blue color. The results are shown in Table 4.

The operation method comprises the following steps: placing the staining solution A and the staining solution B in a 1mL centrifuge tube according to the ratio of 3:7 (300. mu.l: 700. mu.l), shaking up, and recording as a staining solution AB for later use; meanwhile, treating the secretion clinical sample by using a cell preservation solution for later use; the staining solution AB and the clinical sample are uniformly mixed according to the proportion of 1:4, 20ul of the staining solution is placed on a glass slide, a cover glass is covered, and the cell staining condition is observed under a microscope under 40 times of ocular lenses.

Table 4 dyeing liquor accuracy evaluation data of example 6

The detection method comprises the following steps: using the staining solution of example 9, 5 random samples of the secretion were tested to stain each type of cell in vaginal secretion without staining cytoplasm or with light pink or red color and staining nuclei with deep red color. The results are shown in Table 5.

Table 5 dyeing liquor accuracy evaluation data of example 9

Stability evaluation

The staining solutions of examples 1-10 were tested to be stable for at least 12 months without any change in performance when stored at 2-8 ℃.

In conclusion, the staining solution for the visible components of the vaginal secretion is suitable for being used on GMD-S600 full-automatic gynecological secretion analysis system equipment independently developed by Dirui corporation, and is mainly used for being matched with the flow type imaging principle of the GMD-S600 full-automatic gynecological secretion analysis system, so that the integrity, the accuracy and the reliability of imaging of an instrument morphology detection system are guaranteed, an image is real, effective, clear and identifiable, the cell nucleus is uniformly stained, the distribution is staggered, and the staining solution is a necessary reagent for guaranteeing the precision of the visible component detection of a visible component analyzer.

It should be understood that the above embodiments are only examples for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither necessary nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. The stable vaginal secretion visible component staining solution is characterized by comprising a staining solution A and a staining solution B;

2. The stable vaginal secretion visible component staining solution as claimed in claim 1, wherein the organic solvent is one or more of methanol, ethanol, ethylene glycol, propanol, glycerol, butanol, pentanol, acetone, acetonitrile, N-dimethylformamide, and dimethylsulfoxide.

3. The stable vaginal discharge visible component staining solution of claim 1,

the red system coloring agent is one or a mixture of more of neutral red, eosin G, eosin Y, thiazine dyes, Sudan III, red poplar, red tangerine G and hematoxylin;

in the dyeing liquid A, the concentration of the dyeing agent is 0.5-2.0 wt%.

4. The stable vaginal secretion visible component staining solution as claimed in claim 1, wherein the buffer is one or more of HEPES buffer, PBS buffer, MOPSO buffer, BTIS buffer, and Tris buffer, the pH of the buffer is 5.0-9.0, and the concentration of the buffer is 10mM-100 mM.

5. The stable coloring solution for visible components of vaginal secretion according to claim 1, wherein the preservative is one or more of sodium azide, thimerosal, gentamicin, ProClin300, 2-methyl-4-isothiazolin-3-one, sodium pyrithione, and sodium dehydroacetate, and the concentration of the preservative is 0.1-0.75 wt%.

6. The stable vaginal secretion visible component staining solution as claimed in claim 1, wherein the dyeing auxiliary is one or more selected from calcium acetate, sodium acetate, amine acetate, potassium acetate, and magnesium acetate, and the concentration of the dyeing auxiliary is 10mM-100 mM.

7. The stable vaginal secretion tangible element staining solution of claim 1, wherein the osmolality adjusting agent is sodium chloride or potassium chloride and the concentration of the osmolality adjusting agent is 0.5 wt% to 1.5 wt%.

8. The stable coloring solution for visible components of vaginal secretion according to claim 1, wherein the surfactant is one or more selected from polyoxyethylene lauryl ether, Triton X-100, BRIJ35, BRIJ98, EMULGEN24B, span 60, Tween 20, Tween 40, poloxamer, triton X-100, and octylphenol ethoxylate, and the concentration of the surfactant is 0.01 wt% to 1.00 wt%.

9. The stable vaginal secretion tangible composition staining solution of claim 1, wherein the pH of stain a and stain B are each independently 6.0-7.0(25 ± 1) ° c; the electric conductivities are respectively and independently 7.00 +/-0.20 mS/cm (25 +/-1) DEG C; the osmotic pressures were 185. + -.10 mmol/L (mOsm/kg), respectively.

10. The method for preparing a stable coloring solution for visible components of vaginal secretion according to claims 1-9, wherein the components of the coloring solution B are weighed according to the composition, stirred uniformly, adjusted to pH 6.50 ± 0.50(25 ± 1 ℃) by acetic acid or 1M sodium hydroxide, adjusted to osmotic pressure and conductivity, and filtered to obtain the coloring solution B;