CN111721950A - Stable formed component analysis coking liquid and preparation method thereof - Google Patents
Stable formed component analysis coking liquid and preparation method thereof Download PDFInfo
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Abstract
The invention discloses a stable formed component analysis focusing liquid and a preparation method thereof, belonging to the technical field of in-vitro diagnosis of medical instruments. The focusing solution comprises a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent, a dyeing regulator and red blood cell simulation particles, and is mainly used for matching with a focusing function before sample detection of a 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 ensured, the formed image is real, effective, clear and recognizable, the cell morphology distribution is reasonable and well-defined, and the focusing solution is a necessary reagent for ensuring the precision of detecting a visible component by a visible component analyzer.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis of medical instruments, in particular to a stable formed component analysis focusing liquid and a preparation method thereof.
Background
Vaginal secretions, commonly known as "leucorrhea," are fluid secreted by the female reproductive system and mainly consist of a mixture of various secretions such as vaginal mucus, cervical glands, vestibular glands, endometrium, and the like, the formation of which is related to the action of estrogen. Normally, the quality and quantity of the leucorrhea vary with the menstrual cycle. After menstruation, the white leucorrhea is small, white and pasty. When the ovary is about to ovulate in the middle period of menstruation, the cervical gland is exuberant in secretion, the leucorrhea is increased, and the ovary is transparent, slightly sticky and like egg white. After 2-3 days of ovulation, the leucorrhea became turbid, thick, sticky and small in volume. Before and after menstruation, vaginal mucosa exudate is increased and leucorrhea is increased due to pelvic congestion.
In medical clinical examination, microscopy is the most common method, and the examination target is placed under a microscope, and important reference information is provided for medical diagnosis by examining cells, microorganisms or other tangible components in the target. The visible components in the leucorrhea include leucocytes, epithelial cells, bacilli, cocci and the like, and the quantity and the state of the visible components in the leucorrhea can be used as judgment conditions for judging whether the female vagina is healthy or not, and can be used as bases for clinical diagnosis, vaginitis treatment, pedestrian flow, intrauterine device taking and the like. It is known that when abnormal leucorrhea occurs, if timely treatment is not taken, the female can be seriously affected.
With the continuous innovation of technology and the development of leucorrhea microscopic examination, which is one of the current clinical routine laboratory examination projects, the pathological changes of female vagina are reflected by various biological cell conditions in leucorrhea. At present, the inspection of most hospitals to the leucorrhea all adopts the artifical range estimation of microscope, combines chemical reagent to inspect simultaneously, and artifical range estimation is higher to inspection doctor's requirement, and doctor's subjective judgement can the mistake hardly exempted from. Over the years, with the advancement of digital image processing technology, many fields begin to use digital image processing, but the medical field is no exception, and digital image processing brings substantial changes to medical diagnosis, thereby promoting the revolution of medical diagnosis. With the continuous development of intelligent identification technology, the analysis and identification of the visible components in the leucorrhea are gradually started in medical diagnosis, and the use frequency of a medical microscope is reduced as much as possible, so that the eye fatigue strength of medical inspectors can be reduced, and the diagnosis process of doctors can be accelerated. Therefore, the market demand for developing an instrument and a matching reagent capable of rapidly detecting female vaginal secretions is urgent.
According to market demands, a GMD-S600 full-automatic gynecological secretion analysis system is introduced by individual companies, applicable machine types include but are not limited to the above machine types, accurate identification of visible components in a leucorrhea microscopic image is really realized by combining with a matched reagent, and an automatic identification algorithm and related technologies are utilized to replace the most commonly used manual microscopic examination technology at present. 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 cell morphology classification
At present, most of small hospitals in China still use the traditional microscope for detection in leucorrhea detection and then carry out manual treatment. The traditional leucorrhea detection method is to make a sample into a smear, observe basic characteristics of cells and formed components on the smear, such as shape, size and the like, by using a microscope, judge the cleanliness of the formed components according to the result, and diagnose by medical staff according to the judgment result of the leucorrhea cleanliness. The manual detection is greatly influenced by human factors, so that the manual detection 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. The automatic biological cell recognition system for leucorrhea 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, and has very important social value and practical significance for the research of the automatic recognition technology of the biological cell medical microscopic image of leucorrhea.
Disclosure of Invention
The invention aims to provide long-acting stable focusing liquid for analyzing visible components, the effective period can reach 8 months, and the problems that most instruments on the market have no reliable imaging basis before the detection of the visible components are solved.
In order to solve the problems, the invention provides a stable focusing liquid for analyzing a visible component, which comprises a focusing liquid matrix and red blood cell simulation particles, wherein the focusing liquid matrix is prepared from the following components in concentration ratio:
buffer, 10mM-20 mM;
0.7-0.9% of osmotic pressure regulator;
0.01% -1.0% of surfactant;
0.01 to 2.0 percent of preservative;
0.01% -2.0% of chelating agent;
0.01-0.5% of dyeing regulator.
Preferably, the buffer solution is one or a mixture of HEPES, PBS, MOPSO or Tris buffer solution.
Preferably, the osmotic pressure regulator is a mixture of sodium chloride and potassium chloride, wherein the concentration of the sodium chloride is 0.5-1.5%.
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 and octylphenol polyoxyethylene ether.
Preferably, the preservative is one or a mixture of more of sodium azide, thimerosal, gentamicin, ProClin300, sodium pyrithione and sodium dehydroacetate.
Preferably, the chelating agent is one or a mixture of ethylene diamine, 2' -bipyridine, 1, 10-phenanthroline, oxalic acid and ethylenediamine tetraacetic acid.
Preferably, the dyeing regulator is one or a mixture of nitrile yellow, sunset yellow, lemon yellow and methyl orange.
Preferably, the erythrocyte simulation particles are selected from one or more of erythrocytes in bovine blood, sheep blood, pig blood, chicken blood, rabbit blood and dog blood.
A preparation method of stable formed component analysis coking liquid comprises the following steps:
the method comprises the following steps: sequentially weighing raw materials of the coking liquid matrix by using a balance;
step two: adding purified water into a preparation container, sequentially adding the raw materials of the coking liquid matrix weighed in the step one, stirring until the raw materials are completely dissolved, completing the preparation of the coking liquid matrix and continuing stirring;
step three: filtering the coking liquid matrix obtained in the step two, adding red blood cell simulation particles to complete preparation of visible components, and performing split charging inspection according to product specifications;
step four: and after the inspection is reported, the magnetic stirrer is always in a stirring state until the subpackaging is finished, and a product is prepared.
Preferably, the raw materials in the second step are sequentially added and stirred until the raw materials are completely dissolved, and one raw material is required to be completely dissolved and then the next raw material is added; stirring for 20-30min after the coking liquid matrix is obtained, adjusting the pH value to 7.10 +/-0.15, the temperature to 25 +/-1 ℃, the conductivity rho to 15.00 +/-0.50 mS/cm and the osmotic concentration to 270 +/-5.0 mmol/L after stirring; filtering the focusing liquid matrix in the third step by using a 0.22-0.38 mu m polyethylene filter membrane; the concentration of the focusing solution after the red blood cell simulation particles are added is 2850-3250/mu L.
The invention has the advantages of
1. The invention improves the stability of cells, and a surfactant is introduced into formed segregation and focusing liquid to uniformly disperse the cells. The surfactant is active on the surface and the interface, and has extremely high capacity and efficiency of reducing surface tension and interfacial tension. The surfactant has the functions of wetting, dispersing, emulsifying, solubilizing, moisturizing, permeating, preserving corrosion and the like.
2. The invention strictly controls the influence of the matrix on the osmotic pressure of the cells, and the matrix is added with basic components simulating secretion, and the osmotic pressure regulator and various ion components are screened out in an optimal proportion by adopting an orthogonal experiment, thereby ensuring that the osmotic pressure is in a proper range and not influencing the stability of the cells.
3. The dyeing regulator used in the invention is mainly used for making the color appearance of the focusing solution more striking, easily and visually identifying the sedimentation degree of cells and ensuring that the product needs to be fully mixed before use.
4. The invention adopts effective substitutes to simulate the red blood cells in the vaginal secretion, has easily obtained raw materials and stable and accurate evaluation effect, and can realize the market demands of batch production and comprehensive popularization.
Drawings
Fig. 1 shows tangible recognition under a focus interface of a GMD-S600 full-automatic gynecological analysis system.
Detailed Description
The invention provides a stable focusing liquid for visible component analysis, which comprises a focusing liquid matrix and red blood cell simulation particles, wherein the focusing liquid matrix is prepared from the following components in parts by concentration:
buffer, 10mM-20 mM;
0.7-0.9% of osmotic pressure regulator;
0.01% -1.0% of surfactant;
0.01 to 2.0 percent of preservative;
0.01% -2.0% of chelating agent;
0.01-0.5% of dyeing regulator.
Preferably, the buffer solution is one or a mixture of HEPES, PBS, MOPSO or Tris buffer solution; wherein, the preferable pH value is 5.0-9.0, and the concentration is 10mM-20mM PBS buffer solution; most preferred is PBS buffer at a pH of 6.0-8.0 and a concentration of 10 mM.
Preferably, the osmotic pressure regulator is a mixture of sodium chloride and potassium chloride, wherein the concentration of the sodium chloride is 0.5% -1.5%; among them, the concentration of sodium chloride is preferably 0.7% to 0.9%, and the most preferably 0.9%.
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 and octylphenol polyoxyethylene ether; wherein, the concentration is preferably 0.01 to 1.00 percent of triton X-100, and the concentration is most preferably 0.05 percent of triton X-100.
Preferably, the preservative is one or a mixture of more of sodium azide, thimerosal, gentamicin, ProClin300, sodium pyrithione and sodium dehydroacetate; among them, ProClin300 at a concentration of 0.01% to 2.00% is preferable, and ProClin300 at a concentration of 0.03% is most preferable.
The chelating agent is one or a mixture of more of ethylenediamine, 2' -bipyridine, 1, 10-diazophenanthrene, oxalamic acid and ethylenediamine tetraacetic acid; among them, ethylenediaminetetraacetic acid with a concentration of 0.01% to 2.00% is preferable, and ethylenediaminetetraacetic acid with a concentration of 0.05% is most preferable.
Preferably, the dyeing regulator is one or a mixture of nitrile yellow, sunset yellow, lemon yellow and methyl orange; of these, sunset yellow is preferably present at a concentration of 0.01 to 0.50%, and most preferably 0.075%.
Preferably, the erythrocyte simulation particles are selected from one or more of erythrocytes in bovine blood, sheep blood, pig blood, chicken blood, rabbit blood and dog blood; wherein, the porcine whole blood is preferred, the adding amount is preferably 2850-3250/μ L, and the most preferred adding amount is 2950-3180/μ L;
a preparation method of stable formed component analysis coking liquid comprises the following steps:
the method comprises the following steps: sequentially weighing raw materials of the coking liquid matrix by using a balance;
step two: adding purified water into a preparation container, sequentially adding and stirring the raw materials of the coking liquid matrix weighed in the step one until the raw materials are completely dissolved, adding the next raw material after one raw material is required to be completely dissolved in the dissolving process, completing the preparation of the coking liquid matrix and continuously stirring for 20-30min, and adjusting the pH value to 7.10 +/-0.15, the temperature to 25 +/-1 ℃, the conductivity rho to 15.00 +/-0.50 mS/cm and the osmotic concentration to 270 +/-5.0 mmol/L after stirring;
step three: filtering the focusing liquid matrix obtained in the step two by adopting a polyethylene filter membrane of 0.22-0.38 mu m, adding red blood cell simulation particles to finish the preparation of visible components, and performing split charging inspection according to the product specification;
step four: and after the inspection is reported, the magnetic stirrer is always in a stirring state until the subpackaging is finished, and a product is prepared.
Example 1
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 2) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 20min, regulating the pH value to 7.25 by using a pH meter, regulating the conductivity rho to 14.5mS/cm by using a conductivity meter, regulating the osmotic concentration to 265mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 24 ℃; filtering the obtained focusing liquid matrix with 0.22 μm polyethylene filter membrane, adding red blood cell simulation particles with concentration of 3000/μ L to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 2 example 1 formulation ingredients
Example 2
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the dosage of each component is shown in table 3) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 30min, regulating the pH value to 6.95 by using a pH meter, regulating the conductivity rho to 15.5mS/cm by using a conductivity meter, regulating the osmotic concentration to 275mmol/L by using an osmotic pressure meter, and keeping the temperature at about 26 ℃; filtering the obtained focusing liquid matrix with 0.38 μm polyethylene filter membrane, adding 3100/μ L erythrocyte simulated particles to complete preparation of visible components, subpackaging according to product specification, and stirring with magnetic stirrer after inspection to complete subpackaging.
Table 3 example 2 formulation ingredients
Example 3
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 4) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 25min, regulating the pH value to 7.1 by using a pH meter, regulating the conductivity rho to 15mS/cm by using a conductivity meter, regulating the osmotic concentration to 270mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 25 ℃; filtering the obtained focusing liquid matrix with 0.3 μm polyethylene filter membrane, adding red blood cell simulation particles with concentration of 3000/μ L to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 4 example 3 formulation ingredients
Example 4
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 5) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 20min, regulating the pH value to 7.1 by using a pH meter, regulating the conductivity rho to 15mS/cm by using a conductivity meter, regulating the osmotic concentration to 270mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 25 ℃; filtering the obtained focusing liquid matrix with 0.25 μm polyethylene filter membrane, adding red blood cell simulation particles with concentration of 3000/μ L to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 5 example 4 formulation ingredients
Example 5
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 6) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 22min, regulating the pH value to 7.1 by using a pH meter, regulating the conductivity rho to 15mS/cm by using a conductivity meter, regulating the osmotic concentration to 270mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 25 ℃; filtering the obtained focusing liquid matrix with 0.35 μm polyethylene filter membrane, adding 3200/μ L erythrocyte simulation particles to complete preparation of visible components, subpackaging according to product specification, and stirring with magnetic stirrer after inspection to complete subpackaging.
Table 6 example 5 formulation ingredients
Example 6
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 7) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 30min, regulating the pH value to 7.25 by using a pH meter, regulating the conductivity rho to 14.5mS/cm by using a conductivity meter, regulating the osmotic concentration to 265mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 24 ℃; filtering the obtained focusing liquid matrix with 0.22 μm polyethylene filter membrane, adding 3150/μ L erythrocyte simulation particles to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 7 example 6 formulation ingredients
Example 7
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the dosage of each component is shown in table 3) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 30min, regulating the pH value to 6.95 by using a pH meter, regulating the conductivity rho to 15.5mS/cm by using a conductivity meter, regulating the osmotic concentration to 275mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 26 ℃; filtering the obtained focusing liquid matrix with 0.22 μm polyethylene filter membrane, adding 3120/μ L erythrocyte simulated particles to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 8 example 7 formulation ingredients
Example 8
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 4) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 25min, regulating the pH value to 7.1 by using a pH meter, regulating the conductivity rho to 15mS/cm by using a conductivity meter, regulating the osmotic concentration to 270mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 25 ℃; filtering the obtained focusing liquid matrix with 0.28 μm polyethylene filter membrane, adding 3120/μ L erythrocyte simulated particles to complete preparation of visible components, packaging and inspecting according to product specification, and stirring with magnetic stirrer until packaging is completed to obtain the final product.
Table 9 example 8 formulation ingredients
Example 9
Preparing a 10-ten-thousand-level electronic balance, and checking whether the ten-thousand-level balance is qualified in correction; if not, recalibrating is needed; sequentially weighing raw materials of the focusing liquid matrix reagent according to the formula composition, and then accurately weighing by using a universal balance; adding a full batch of purified water into a clean high-density polyethylene bottle, sequentially adding a buffer solution, an osmotic pressure regulator, a surfactant, a preservative, a chelating agent and a dyeing regulator (the use amounts of all the components are shown in table 4) into the high-density polyethylene bottle in sequence, adding the next raw material after one raw material is completely dissolved, stirring the mixture until the raw material is completely dissolved to obtain a coking liquid matrix, continuously stirring the mixture for 30min, regulating the pH value to 7.2 by using a pH meter, regulating the conductivity rho to 15.3mS/cm by using a conductivity meter, regulating the osmotic concentration to 272mmol/L by using an osmotic pressure meter, and simultaneously keeping the temperature at about 25 ℃; filtering the obtained focusing liquid matrix with 0.38 μm polyethylene filter membrane, adding red blood cell simulation particles with concentration of 3100/μ L to complete preparation of visible components, subpackaging according to product specification, and stirring with magnetic stirrer after inspection until subpackaging is completed to obtain the final product.
Table 10 example 9 formulation ingredients
Example 10
The finished product is obtained after the formula is prepared according to the above embodiments, and the performance of the formed component analysis coking liquid obtained in the embodiments 1, 3, 6 and 9 is evaluated by the following specific method:
1. visual inspection of appearance
In each of the focusing liquids for analyzing the visible components obtained in examples 1, 3, 6 and 9, 5 bottles of the focusing liquid for analyzing the visible components were randomly selected to check the appearance performance index by normal vision, and the following requirements were satisfied: the coking liquid is a slightly mixed orange suspension. The appearance results of 5 bottles are randomly extracted and visually observed to be qualified.
2. Focus test
In examples 1, 3, 6 and 9, 10 bottles of the focusing solution for analyzing the visible components were taken from each group of the focusing solutions for analyzing the visible components and were subjected to focusing test in a GMD-S600 full-automatic gynecological secretion analysis system, and the test results should show a passing status after each bottle of the focusing solution was tested. The test results are all in a qualified state.
(results are shown in tables 11-14)
TABLE 11 Focus test results data for finished products obtained in example 1
Table 12 data of focus test results for finished products obtained in example 3
TABLE 13 Focus test results data for finished products obtained in example 6
TABLE 14 Focus test results data for finished products obtained in example 9
3. Stability of expiry date
The formed component analysis focusing liquid obtained in example 1, example 3, example 6 and example 9 is taken, under the storage condition specified at 2-8 ℃, the focusing liquid obtained one month (9 months) after the expiration date is continuously focused 10 times on a GMD-S600 full-automatic gynecological secretion analysis system, and the test result shows that the focusing liquid passes the condition. The test results are all in a qualified state. (results are shown in tables 15-19)
TABLE 15 test value data for the focusing fluid (9 months) of example 1
TABLE 16 test value data for the focusing fluid (9 months) of example 3
TABLE 17 test data for the focusing fluid (9 months) in example 6
TABLE 18 data from the test of the Focus solution (9 months) of example 9
The performance evaluation test results show that the evaluation results are qualified, the focusing liquid has stable quality, the cell morphology is good, the dispersion is uniform, the implementation cases can effectively focus on the optical unit of the GMD-S600 full-automatic gynecological secretion analysis system, and the evaluation results are excellent; taking the formulation example 1 as an example, as shown in fig. 1, the cells are uniformly dispersed under the mirror, the cell morphology is full and uniform, the adhesion condition between the cells is avoided, the accuracy of the cell counting function under the test condition is facilitated, the clear and accurate imaging of the cells can be ensured, and the series of formulations are all designed formulations with excellent cell morphology, high accuracy and good stability.
In conclusion, the visible component analysis focusing liquid can be stored at 2-8 ℃ for at least 8 months stably without changing the performance, is suitable for GMD-S600 full-automatic gynecological secretion analysis system, is suitable for machine types including but not limited to the machine types, is used on equipment, does not have products of the type on the market at present, is reliable and original in independent research and development process, is mainly used for matching with the focusing function before sample detection of the GMD-S600 full-automatic gynecological secretion analysis system, so that the imaging integrity, the imaging accuracy and the imaging reliability of an instrument morphology detection system are ensured, images are real, effective, clear and identifiable, the cell morphology distribution is reasonable and wrong, and the focusing function is a necessary reagent for ensuring the visible component analyzer to detect the visible components, the invention is not limited to the examples, and can be improved or changed according to the description by a person skilled in the art, all such modifications and variations are intended to be included herein within the scope of this disclosure and the appended claims.
Claims (10)
1. The stable focusing liquid for analyzing the visible components is characterized by comprising a focusing liquid matrix and red blood cell simulation particles, wherein the focusing liquid matrix is prepared from the following components in percentage by concentration:
buffer, 10mM-20 mM;
0.7-0.9% of osmotic pressure regulator;
0.01% -1.0% of surfactant;
0.01 to 2.0 percent of preservative;
0.01% -2.0% of chelating agent;
0.01-0.5% of dyeing regulator.
2. The stabilized focusing solution for forming ingredient analysis according to claim 1, wherein the buffer is one or a mixture of HEPES, PBS, MOPSO or Tris buffer.
3. The stable, tangible, analytical focusing fluid of claim 1 wherein the osmolality adjusting agent is a mixture of sodium chloride and potassium chloride, wherein the sodium chloride is present in a concentration of 0.5% to 1.5%.
4. The stable char forming solution for analyzing physical components according to claim 1, wherein the surfactant is one or a mixture of polyoxyethylene lauryl ether, Triton X-100, BRIJ35, BRIJ98, EMULGEN24B, span 60, Tween 20, Tween 40, poloxamer, and octylphenol ethoxylate.
5. The stabilized focusing fluid for forming ingredient analysis according to claim 1, wherein the preservative is one or more selected from the group consisting of sodium azide, thimerosal, gentamicin, ProClin300, sodium pyrithione, and sodium dehydroacetate.
6. The stabilized char forming fraction focusing solution according to claim 1, wherein the chelating agent is one or more selected from the group consisting of ethylenediamine, 2' -bipyridine, 1, 10-diazophane, oxalamic acid, and ethylenediaminetetraacetic acid.
7. The stabilized focusing fluid for analyzing formed components according to claim 1, wherein the dyeing regulator is one or more selected from nitrile yellow, sunset yellow, lemon yellow, and methyl orange.
8. The stabilized focusing solution for forming ingredient analysis according to claim 1, wherein the red blood cell-simulating particles are selected from one or more red blood cells in bovine blood, sheep blood, pig blood, chicken blood, rabbit blood, and dog blood.
9. The method of claim 1, comprising the steps of:
the method comprises the following steps: sequentially weighing raw materials of the coking liquid matrix by using a balance;
step two: adding purified water into a preparation container, sequentially adding the raw materials of the coking liquid matrix weighed in the step one, stirring until the raw materials are completely dissolved, completing the preparation of the coking liquid matrix and continuing stirring;
step three: filtering the focusing liquid matrix obtained in the step two, adding red blood cell simulation particles to complete the preparation of the visible components, and performing split charging inspection according to the product specification;
step four: and after the inspection is reported, the magnetic stirrer is always in a stirring state until the subpackaging is finished, and a product is prepared.
10. The method of claim 9, wherein the raw materials are sequentially added and stirred until completely dissolved in step two, and one raw material is required to be completely dissolved before the next raw material is added; stirring for 20-30min after the coking liquid matrix is obtained, adjusting the pH value to 7.10 +/-0.15, the temperature to 25 +/-1 ℃, the conductivity rho to 15.00 +/-0.50 mS/cm and the osmotic concentration to 270 +/-5.0 mmol/L after stirring; filtering the focusing liquid matrix in the third step by using a 0.22-0.38 mu m polyethylene filter membrane; the concentration of the focusing solution after the red blood cell simulation particles are added is 2850-3250/mu L.
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