CN107167356B - Coloring agent capable of quickly developing color after cells in urine are colored and using method thereof - Google Patents
Coloring agent capable of quickly developing color after cells in urine are colored and using method thereof Download PDFInfo
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- CN107167356B CN107167356B CN201710207744.4A CN201710207744A CN107167356B CN 107167356 B CN107167356 B CN 107167356B CN 201710207744 A CN201710207744 A CN 201710207744A CN 107167356 B CN107167356 B CN 107167356B
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- 210000002700 urine Anatomy 0.000 title claims abstract description 92
- 238000000034 method Methods 0.000 title claims abstract description 44
- 239000003086 colorant Substances 0.000 title claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 134
- 238000010186 staining Methods 0.000 claims abstract description 73
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 57
- 210000004027 cell Anatomy 0.000 claims abstract description 55
- 239000000975 dye Substances 0.000 claims abstract description 40
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 22
- 239000002904 solvent Substances 0.000 claims abstract description 22
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000008139 complexing agent Substances 0.000 claims abstract description 13
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- 239000001044 red dye Substances 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims description 61
- 238000004043 dyeing Methods 0.000 claims description 52
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- 239000008213 purified water Substances 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 14
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid group Chemical group C(CC(O)(C(=O)O)CC(=O)O)(=O)O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 12
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- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 claims description 5
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- PDIDTMXYILXCLW-UHFFFAOYSA-M sodium;propanoate;propanoic acid Chemical compound [H+].[Na+].CCC([O-])=O.CCC([O-])=O PDIDTMXYILXCLW-UHFFFAOYSA-M 0.000 claims description 5
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- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 4
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 4
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- 239000000203 mixture Substances 0.000 claims description 4
- 229910052700 potassium Inorganic materials 0.000 claims description 4
- BUCIWTBCUUHRHZ-UHFFFAOYSA-K potassium;disodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O BUCIWTBCUUHRHZ-UHFFFAOYSA-K 0.000 claims description 4
- LJSOLTRJEQZSHV-UHFFFAOYSA-L potassium;sodium;hydron;hydroxide;phosphate Chemical compound [OH-].[Na+].[K+].OP(O)([O-])=O LJSOLTRJEQZSHV-UHFFFAOYSA-L 0.000 claims description 4
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- 229910052708 sodium Inorganic materials 0.000 claims description 4
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- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 4
- OAOXWLMNOXWELO-UHFFFAOYSA-N [methyl-[3-[methyl(trihydroxymethyl)amino]propyl]amino]methanetriol Chemical compound OC(O)(O)N(C)CCCN(C)C(O)(O)O OAOXWLMNOXWELO-UHFFFAOYSA-N 0.000 claims description 3
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
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- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims 2
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- MCHZKGNHFPNZDP-UHFFFAOYSA-N 2-aminoethane-1,1,1-triol;hydrochloride Chemical compound Cl.NCC(O)(O)O MCHZKGNHFPNZDP-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
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- DIBSKMYGUIFFND-UHFFFAOYSA-N [3-[hydroxymethyl(methyl)amino]propyl-methylamino]methanol Chemical compound OCN(C)CCCN(C)CO DIBSKMYGUIFFND-UHFFFAOYSA-N 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
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- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 1
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- 229910052740 iodine Inorganic materials 0.000 description 1
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- 239000012528 membrane Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- YDXZZRPYFRMWEP-UHFFFAOYSA-N methanol;propane-1,2,3-triol;hydrate Chemical compound O.OC.OCC(O)CO YDXZZRPYFRMWEP-UHFFFAOYSA-N 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
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- 230000010399 physical interaction Effects 0.000 description 1
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229940099373 sudan iii Drugs 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
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- 230000002485 urinary effect Effects 0.000 description 1
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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
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a staining agent for rapid color development after staining cells in urine and a using method thereof, wherein the staining agent comprises a reagent A and a reagent B, the mass ratio of the reagent A to the reagent B is 0.25-4, and in the staining agent, the reagent A comprises neutral red dye 0.6-1.2 g; 100g of dye solvent; the reagent B comprises 10 mM-600 mM of buffering agent; 0.1-8 g of cell membrane damaging agent; 0-4 g of complexing agent; 0.01-10 g of pH regulator; 100g of solvent. The staining agent can directly stain cells in urine without pretreating the cells, and has the advantages of short color development time, simple operation and high detection efficiency; in addition, the coloring agent does not influence the morphology of the red blood cells with pathological significance, does not cause the aggregation of a high-concentration red blood cell sample, is favorable for improving the accuracy of a detection result, and can realize automatic detection.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a staining agent capable of quickly developing color after staining cells in urine and a using method thereof.
Background
The urine contains various components such as red blood cells, white blood cells, casts, and epithelial cells. The analysis of the visible components is of great significance for the detection of diseases of the urinary system, the adjacent reproductive system and other systems. A large number of researches show that crystals and saccharomycetes in urine interfere with analysis of urine red blood cells, a large number of mucus filaments and hyphae can cause false positive of cast counting, white blood cells, small phagocytes, and white blood cells, large red blood cells and white blood cells with unobvious cell nucleuses, have similar shapes with trichomonas, spores and red blood cells, and transparent casts and sperms are colorless, so that the probability of false detection and missed detection is high, and the chemical dyeing has great significance for identifying pathological particles. Although the existing urine has more dyeing methods for visible components, the existing urine has the defects, such as Sternheimer dyeing method, Sternheimer-Malbin dyeing method, Burhe-Muhlberg dyeing method and Aliskiron-neutral red dyeing method, the dyeing time is long (>1 minute), the coloring of homogeneous cells is not uniform, the dyeing effect is limited by the pH value of a urine sample, and in addition, the composite dye is easy to generate particle interferents during dyeing; the sample preparation process of fixed dyeing methods such as May-Giemsa, Raynaud, Gieser and Raymi composite dyeing methods is complex, and is not beneficial to improving the detection speed and realizing the purpose of automatic detection; the special dyeing methods such as Sudan III dyeing method, iodine dyeing method, peroxidase dyeing method, and hemoxanthin-containing dyeing method are only suitable for analyzing a certain type of visible components in urine samples.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a staining agent for rapid color development after staining cells in urine and a using method thereof, and aims to solve the problems that the existing urine cell staining method is complex in sample preparation process, long in staining time, easy to aggregate red blood cell morphology due to the influence of staining solution, and not beneficial to automatic detection.
The technical scheme of the invention is as follows:
a staining agent capable of quickly developing color after staining cells in urine comprises a reagent A and a reagent B, wherein the mass ratio of the using amounts of the reagent A to the reagent B is 0.25-4, and in the staining agent,
the reagent A comprises:
0.6-1.2 g of neutral red dye;
100g of dye solvent;
the reagent B comprises:
the staining agent capable of rapidly developing color after staining cells in urine is prepared by using a solvent, wherein the solvent is one or more of purified water and an organic solvent.
The staining agent capable of rapidly developing color after staining cells in urine is characterized in that the organic solvent can be one or a combination of methanol, ethanol, ethylene glycol, amyl alcohol, glycerol and dimethyl sulfoxide.
The staining agent capable of rapidly developing color after staining cells in urine is a mixture of one or more of 2- (N-morpholine) ethanesulfonic acid, 1, 3-di [ (trihydroxymethyl) methylamino ] propane, piperazine-1, 4-diethylsulfonic acid, 3- (N-morpholinyl) propanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, trihydroxymethyl aminomethane-hydrochloric acid, barbituric acid, propionic acid-sodium propionate and acetic acid-sodium acetate.
The staining agent capable of rapidly developing color after staining cells in urine is characterized in that the cell membrane damaging agent is one or more of the following organic compounds:
CH3(CH2)n1COOR，n10, 1, 2 or 3, wherein R is Na, H or K;
the staining agent capable of rapidly developing color after staining cells in urine is characterized in that the complexing agent is citric acid, citrate or ethylenediamine tetraacetate.
A method for using a staining agent capable of rapidly developing color after staining cells in urine comprises the following steps:
step S1, preparing a reagent A and a reagent B;
step S2, mixing the prepared reagent A and the prepared reagent B according to the mass ratio of 0.25-4 to obtain a coloring agent, and then dropwise adding the coloring agent to a concentrated or non-concentrated urine sample;
and step S3, dyeing for 5-30S to obtain a dyed urine sample.
The use method of the staining agent capable of rapidly developing color after staining cells in urine comprises the following steps of:
preparation of reagent A:
a1, adding a dye solvent and a chemical dye into a reagent bottle in the order to obtain a first mixed solution A1;
a2, placing the reagent bottle containing the first mixed solution A1 on a shaking table to be fully dissolved for 2-5 hours to obtain a second mixed solution A2;
A. filtering the second mixed solution A2, marking the filtrate as a reagent A, and storing for later use;
preparation of reagent B:
b1, adding a buffering agent, a complexing agent and a cell membrane damaging agent into purified water, and fully dissolving the materials to obtain a first mixed solution B1;
b2, adding a pH regulator into the first mixed solution B1 to regulate the pH value to 4.8-7.0 to obtain a second mixed solution B2;
B. the second mixed solution B2 was filtered, and the filtrate was labeled as reagent B and kept for future use.
The use method of the staining agent capable of rapidly developing the color after staining the cells in the urine comprises the step of using a reagent A and a reagent B in the staining agent according to a mass ratio of 0.25-4.
The use method of the staining agent capable of rapidly developing the color after staining the cells in the urine comprises the step of using the staining agent and the urine sample in a volume ratio of 1-8.
Has the advantages that: the reagent for rapid color development after staining the cells in the urine can directly stain the cells in the urine without pretreating the cells in the urine, and has the advantages of short color development time, simple operation and high detection efficiency; in addition, the coloring agent does not influence the morphology of the red blood cells with pathological significance, does not cause the aggregation of a high-concentration red blood cell sample, and is favorable for improving the accuracy of a detection result and realizing the purpose of automatic detection.
Drawings
Fig. 1, 2 and 12 are graphs showing the effect of the staining agent and the staining method prepared according to the embodiment 4 of the invention on white blood cells in urine.
Fig. 3, 4 and 5 are graphs showing the effect of the staining agent and the staining method prepared in example 1 of the present invention on the epithelial cells (fig. 4 containing clue cells) in urine.
FIGS. 6, 7 and 8 are graphs showing the effect of the staining agent and the staining method prepared in example 2 of the present invention on tube types in urine.
Fig. 9, 10 and 11 are graphs showing the effect of the staining agent and the staining method prepared in example 3 of the present invention on red blood cells in urine.
Detailed Description
The invention provides a staining agent capable of rapidly developing color after staining cells in urine and a using method thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a staining agent capable of rapidly developing color after staining cells in urine, which comprises a reagent A and a reagent B, wherein the mass ratio of the reagent A to the reagent B is 0.25-4, and in the staining agent,
the reagent A comprises:
0.6-1.2 g of neutral red dye;
100g of dye solvent;
the reagent B comprises:
wherein the chemical dye is selected to be an organic dye that is capable of diffusing into the interior of the cell membrane and distinguishing the cytoplasm of the cell nucleus. The way chemical dyes interact with cells is divided into physical interactions, which follow similar compatibility principles, and chemical interactions, which include electrostatic interactions, hydrogen bonding interactions, van der waals forces, hydrophobic forces, and covalent bonds, with electrostatic interactions being the most common and contributing the most to the various forces. The diffusion rate of the dye is affected by the molecular weight of the dye, the higher the molecular weight, the slower the diffusion rate.
Based on the above-mentioned action principle, the chemical dye is preferably a neutral red dye as a weakly basic dye, which is positively charged under near neutral and alkaline conditions and which interacts with the phosphate group on the chromatin molecule containing negatively charged groups in the cell, the carboxyl group on the protein molecule and the sialic acid molecule on the cell membrane to develop red color. The chromatin in the cell nucleus is compact and the color is deep, and the chromatin and protein in the cytoplasm are distributed loosely and the color is light. Various cell types can be clearly distinguished according to the cell morphology after staining. In addition, the dye has wide application range, and can dye various components in urine, such as white blood cells, epithelial cells, red blood cells, sperms and the like. In the invention, the mass fraction of the neutral red dye in the mixed solution of A and B is 0.12-0.96%, and the neutral red dye with proper dosage can rapidly dye cells, and the nucleus and cytoplasm of the dyed cells have clear structures and are easy to distinguish, thereby being beneficial to improving the inspection efficiency.
The solvent is one or more of purified water and an organic solvent, and has the functions of enabling the chemical dye to be stably stored for a long time and enabling the prepared dyeing agent to keep a good dyeing effect. The purified water is beneficial to increasing the solubility of salts, and can dilute the organic solvent to prevent the deformation of red blood cells in the urine sample caused by excessive organic solvent. In addition, the method for determining the amount of purified water needs to satisfy two requirements: one is to ensure that the red blood cells do not deform, and the other is to ensure that the dye solution has no educt. In the invention, the mass fraction of the reagent A in the mixed solution of A and B is 20-80%. The organic solvent of the dye solvent may be methanol, ethanol, ethylene glycol, amyl alcohol, glycerol, dimethyl sulfoxide (DMSO), or a combination thereof. Such as methanol-ethanol, glycol-methanol, DMSO-glycol, glycol-glycerol or a mixture of multiple organic solvents. The organic solvent and water can also be compounded to serve as a dye solvent, such as methanol-water, ethanol-water, glycol-water, amyl alcohol-water, glycerol-water, dimethyl sulfoxide (DMSO) -water, glycol-methanol-water, glycol-ethanol-water, glycol-glycerol-water, and glycerol-methanol-water, preferably, the effect of the glycol-water or the glycol-methanol-water as the dye solvent is better.
The buffer agent has the main functions of adjusting the pH value and osmotic pressure of urine, maintaining the morphology of red blood cells, and simultaneously ensuring a relatively constant dyeing environment so that the dyeing effect of the dye on cells in different urine samples is similar. For urine systems, the buffer is preferably selected to have a pH of 4.8 to 7.0, more preferably 4.8 to 6.8, and most preferably 5.0 to 6.2. The buffer can be one of 2- (N-morpholine) ethanesulfonic acid (MES), 1, 3-Bis [ (Tris (hydroxymethyl) methylamino ] propane (Bis-Tris), piperazine-1, 4-diethylsulfonic acid (PIPES), 3- (N-morpholinyl) propanesulfonic acid (MOPS) and 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), or one of disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, Tris-hydrochloric acid, barbiturate-hydrochloric acid, propionic acid-sodium propionate and acetic acid-sodium acetate, or a composite buffer system among the above buffers, for example, a compound system of 1, 3-Bis [ (hydroxymethyl) methylamino ] propane (Bis-Tris) and propionic acid-sodium propionate is selected, can directly adjust the pH value and osmotic pressure of urine to proper values, and is convenient for the subsequent dyeing process.
When the buffering agent plays a role in adjusting the pH value, the concentration of the buffering agent in the solution obtained by mixing the reagent A and the reagent B is more proper, and the concentration of the buffering agent is 10 mmol/L-600 mmol/L; in order to ensure better dyeing effect, the concentration of the buffering agent in the mixed solution of the reagent A and the reagent B is preferably 10 mmol/L-200 mmol/L.
The membrane damaging agent is an organic compound molecule capable of breaking the barrier of the cell membrane of a living cell, and mainly acts on a phospholipid bilayer, that is, a hydrophobic end of the molecule is embedded into the phospholipid bilayer, so that the phospholipid bilayer is loosely arranged, and thus, the phospholipid bilayer with the increased surface area facilitates the diffusion of small molecules into the interior of the cell, such as dye molecules, which can freely diffuse into the interior of the cell from the damaged cell membrane to stain the nucleus and cytoplasm. At the same time, it is required that the cell membrane damaging substances, when acting on the phospholipid bilayer, do not affect the cell morphology, in particular the morphology of the fragile, diagnostically significant erythrocytes. The dosage of the cell membrane injury substance is determined by that no obvious deformation of red blood cells is generated and the red blood cell count is unchanged when the cell membrane injury substance is observed under a microscope for 5 minutes. The cell membrane damaging agent can be divided into the following three classes:
CH3(CH2)n1COOR, wherein n1 ═ 0, 1, 2 or 3, and R may be Na, H or K; preferably, when n1 is 1 or 2, the cell membrane damaging agent is effective, and when the cell membrane damaging agent is present in a solution in which the reagent a and the reagent B are mixed in an amount of 0.1% to 8% by mass, preferably 1% to 4% by mass, the cell membrane damaging agent can be ensuredOn the premise that the cell morphology with diagnostic significance is not affected, the transmembrane speed of dye molecules is improved, and cells in urine are quickly dyed.
The complexing agent can form a stable water-soluble complex with alkali metal, rare earth element, transition metal and the like, and the main function of the complexing agent is to reduce the interference of amorphous salt in urine on the dyeing process. In the invention, the complexing agent can be citrate or Ethylene Diamine Tetraacetic Acid (EDTA) salt, and the dosage of the complexing agent is that the mass concentration of the solution obtained by mixing the reagent A and the reagent B is preferably 0-4%.
The pH value regulator is used for further regulating the pH value of the dyeing agent, so that the dyeing pH value environment is ensured, the dyeing agent can be used for quickly dyeing cells in urine, and the shape of the urine cannot be influenced. Preferably, the pH regulator may be acetic acid, propionic acid, hydrochloric acid, etc. to adjust the pH of the reagent B to 4.8-7.0.
The solvent B is used for fully dissolving the added salts to obtain a uniform and stable reagent B which is convenient to be matched with the reagent A for application so as to dye cells in urine. In the invention, the solvent in the reagent B is preferably purified water, so that the reagent B has better solubility on different substances added and does not influence cells in urine.
In addition, the invention also provides a use method of the staining agent for rapid color development after staining cells in urine, which comprises the following steps:
step S1, preparing a reagent A and a reagent B;
step S2, mixing the prepared reagent A and the prepared reagent B according to the mass ratio of 0.25-4 to obtain a coloring agent, and then dropwise adding the coloring agent onto the concentrated urine sample;
and step S3, dyeing for 5-30S to obtain a dyed urine sample.
Preferably, the step of preparing reagent a comprises: adding a dye solvent into a reagent bottle, then adding a chemical dye to obtain a first mixed solution A1, placing the reagent bottle containing the first mixed solution A1 on a shaking table to be fully dissolved for 2-5 hours to obtain a second mixed solution A2, filtering the second mixed solution A2, marking the filtrate as a reagent A, and storing for later use. The preparation of reagent B comprises the following steps: adding a buffering agent, a complexing agent and a cell membrane damaging agent into solvent purified water, fully dissolving to obtain a first mixed solution B1, then adjusting the pH value of the first mixed solution B1 to 4.8-7.0 by using a pH regulator to obtain a second mixed solution B2, finally filtering the second mixed solution B2, marking the filtrate as a reagent B, and storing for later use.
And mixing the prepared reagent A and the prepared reagent B according to the mass ratio of 0.25-4 to obtain a coloring agent, and dripping the coloring agent on the non-concentrated or concentrated urine sample. Preferably, the mass ratio of the reagent A to the reagent B is 0.67-1.5, and when the reagent A and the reagent B are mixed according to the above proportion, the dyeing agent solution is more uniform, the property is more stable, and the dyeing speed is higher. The volume ratio of the added coloring agent to the urine sample is 1-8, because when the volume ratio of the urine to the coloring agent is in the range, the coloring agent can quickly and effectively dye cells in the urine, and the cells in the urine cannot be damaged by the added coloring agent to the original structure and form, so that the accuracy of a detection result is improved.
And adding a proper amount of coloring agent into the concentrated urine, and dyeing for 5-30 s to obtain a dyed urine sample. In addition, the staining solution of the method is not only suitable for microscopic examination, but also can be applied to a flow digital image analysis technology, and is beneficial to improving the detection efficiency and the accuracy of a detection result.
Example 1
1. Preparation of staining agent reagent a:
adding 5.00g of methanol, 95.00g of ethylene glycol and 1.20g of neutral red into a brown reagent bottle in sequence to obtain a first mixed solution A1; then, placing the brown reagent bottle filled with the first mixed solution A1 on a shaker, and dissolving at medium speed for 3 hours to completely dissolve the medicine to obtain a second mixed solution A2; the second mixed solution A2 was then filtered through a 0.22um filter, and the filtered solution was labeled reagent A and stored in a brown bottle.
And (3) reagent B:
a first mixed solution B1 was obtained by adding 100.00g of purified water, 4.20g (200mM) of 1, 3-Bis [ (tris (hydroxymethyl) methylamino ] propane (Bis-tris) and 8.00g of sodium propionate to a reagent bottle and dissolving them sufficiently, propionic acid was added to the first mixed solution B1 to adjust the pH of the solution B1 to within the range of 5.50. + -. 0.05 (25. + -. 1 ℃), the second mixed solution B2 was filtered with a 0.22 μm filter, and the filtered solution was labeled as reagent B and stored in the reagent bottle.
2. Dyeing method
The reagent A and the reagent B are mixed according to the mass ratio of 1:1, and are shaken to prepare the ready-to-use dyeing solution. The urine sample can be concentrated by a centrifugal method, the concentrated urine sample is placed in a counting plate, then the prepared staining solution is directly dripped into the concentrated or non-concentrated urine sample, and the proportion of the staining solution to the urine sample is 1: and 8, dyeing for 10s to obtain a dyed urine sample.
Example 2
1. Preparation of staining agent reagent a:
adding 100.00g of ethylene glycol and 1.20g of neutral red into a brown reagent bottle in sequence to obtain a first mixed solution A1; then, placing the brown reagent bottle filled with the first mixed solution A1 on a shaker, and dissolving at medium speed for 3 hours to completely dissolve the medicine to obtain a second mixed solution A2; the second mixed solution A2 was then filtered through a 0.22um filter, and the filtered solution was labeled reagent A and stored in a brown bottle.
And (3) reagent B:
a reagent bottle was filled with 100.00g of purified water, 2.10g (50mM) of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES), 4.00g of sodium propionate, 4.00g of EDTA-K2Fully dissolving to obtain a first mixed solution B1; the pH of the first mixed solution B1 was adjusted to within a range of 6.00 ± 0.05 (25 ± 1 ℃) with propionic acid; the second mixed solution B2 was then filtered through a 0.22 μm filter, and the filtered solution was labeled as reagent B and stored in a reagent bottle.
2. Dyeing method
Mixing the reagent A and the reagent B in a ratio of 0.25: 1, and shaking uniformly to prepare the ready-to-use dyeing solution. Placing the urine sample concentrated by a centrifugal method in a counting plate, and then directly dripping prepared dyeing liquid into the concentrated or non-concentrated urine sample, wherein the volume ratio of the dyeing liquid to the urine sample is 1: and 1, dyeing for 30s to obtain a dyed urine sample.
Example 3
1. Preparation of staining agent reagent a:
adding 100g of glycerol and 0.6g of neutral red into a brown reagent bottle in sequence to obtain a first mixed solution A1; then, placing the brown reagent bottle filled with the first mixed solution A1 on a shaker, and dissolving at medium speed for 3.5 hours to completely dissolve the medicine to obtain a second mixed solution A2; the second mixed solution A2 was then filtered through a 0.22um filter, and the filtered solution was labeled reagent A and stored in a brown bottle.
And (3) reagent B:
adding 100g of purified water, 0.61g (50mM) of Tris (hydroxymethyl) aminomethane (Tris), 8g of sodium propionate and 1.9g of citric acid into a reagent bottle, and fully dissolving to obtain a first mixed solution B1; the pH of the first mixed solution B1 was adjusted to within a range of 6.70 ± 0.05 (25 ± 1 ℃) with propionic acid; the second mixed solution B2 was then filtered through a 0.22 μm filter, and the filtered solution was labeled as reagent B and stored in a reagent bottle.
2. Dyeing method
Mixing the reagent A and the reagent B in a ratio of 4: 1, and shaking uniformly to prepare the ready-to-use dyeing solution. Placing the urine sample concentrated by a centrifugal method in a counting plate, and then directly dripping prepared dyeing liquid into the concentrated or non-concentrated urine sample, wherein the volume ratio of the dyeing liquid to the urine sample is 3: and 7, dyeing for 5s to obtain a dyed urine sample.
Example 4
1. Preparation of staining agent reagent a:
adding 5g of methanol, 95g of ethylene glycol and 0.8 g of neutral red into a brown reagent bottle in sequence to obtain a first mixed solution A1; then, placing the brown reagent bottle filled with the first mixed solution A1 on a shaker, and dissolving at medium speed for 4 hours to completely dissolve the medicine to obtain a second mixed solution A2; the second mixed solution A2 was then filtered through a 0.22um filter, and the filtered solution was labeled reagent A and stored in a brown bottle.
And (3) reagent B:
adding 100g of purified water, 4g of Bis-tris and 8g of sodium propionate into a reagent bottle, and fully dissolving to obtain a first mixed solution B1; the pH value of the first mixed solution B1 was adjusted to be within a range of 5.00 ± 0.05 (25 ± 1 ℃) using propionic acid; the second mixed solution B2 was then filtered through a 0.22 μm filter, and the filtered solution was labeled as reagent B and stored in a reagent bottle.
2. Dyeing method
Mixing the reagent A and the reagent B in a ratio of 3: 2, and shaking up to prepare the ready-to-use dyeing solution. Placing the urine sample concentrated by a centrifugal method in a counting plate, and then directly dripping prepared dyeing liquid into the concentrated or non-concentrated urine sample, wherein the volume ratio of the dyeing liquid to the urine sample is 1: and 6, obtaining a dyed urine sample after dyeing for 20 s.
Example 5
1. Preparation of staining agent reagent a:
adding 5g of methanol, 95g of ethylene glycol and 1.2g of neutral red into a brown reagent bottle in sequence, then placing the brown reagent bottle filled with the first mixed solution A1 on a shaker, and dissolving at medium speed for 5 hours to completely dissolve a medicine to obtain a second mixed solution A2; the second mixed solution A2 was then filtered through a 0.22um filter, and the filtered solution was labeled reagent A and stored in a brown bottle.
And (3) reagent B:
adding 100g of purified water, 3g of Bis-tris and 6g of sodium propionate into a reagent bottle, and fully dissolving to obtain a first mixed solution B1; the pH value of the first mixed solution B1 was adjusted within the range of 5.80. + -. 0.05 (25. + -. 1 ℃ C.) with acetic acid; the second mixed solution B2 was then filtered through a 0.22 μm filter, and the filtered solution was labeled as reagent B and stored in a reagent bottle.
2. Dyeing method
Mixing the reagent A and the reagent B in a ratio of 2: 3, and shaking uniformly to prepare the ready-to-use dyeing solution. Placing the urine sample concentrated by a centrifugal method in a counting plate, and then directly dripping prepared dyeing liquid into the concentrated or non-concentrated urine sample, wherein the volume ratio of the dyeing liquid to the urine sample is 1: and 4, dyeing for 15s to obtain a dyed urine sample.
In the present invention, as can be seen from fig. 1 and 2, the white blood cell nuclei are densely stained with deep red color, the boundary of the nuclei is clear, and the cytoplasm is lightly stained due to relatively less chromatin and protein components.
As can be seen in FIGS. 3, 4 and 5, the nuclei of epithelial cells were stained deep red due to dense chromatin, the boundaries of the nuclei were clear, and the cytoplasm was lightly stained due to relatively small chromatin and protein components.
As can be seen from FIGS. 6 and 7, the tube-shaped surface glycoprotein is lightly stained, and the inclusion substance contains protein components, so that the staining is deep and clearly visible.
As can be seen in FIG. 8, the light staining of the trichomonas, the dye increased the contrast between the sample and the background, so that trichomonas flagella and alveolar nuclei were visible and easily distinguished from the stained leukocytes;
as can be seen in fig. 9, the dye increased the contrast between the sample and the background, and the nuclei and tails of the sperm were clearly visible after staining, readily distinguishable from the unstained yeast.
As can be seen in fig. 10 and 11, the red blood cells in the urine were not stained or lightly stained, and the red blood cells were well defined.
In FIG. 12, it can be seen that the mucus thread and yeast are not stained, but the staining background is slightly colored due to the action of the dye, and the contrast of the photo is deepened, so that the non-stained yeast is easily identified with the white blood cells.
The staining agent capable of rapidly developing color after staining cells in urine and the use method thereof have good staining effect on different cells in urine.
In conclusion, the invention provides the reagent for rapid color development after the staining of the cells in the urine and the use method thereof, the staining agent can rapidly stain the cells in the urine without damaging the morphology of the red blood cells, the use method is simple, the stained cells are uniform in color development, the efficiency and the accuracy of manual microscopic examination can be improved, and the development significance of the flow cell-digital color imaging technology is profound.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (5)
1. A staining agent capable of rapidly developing color after staining cells in urine is characterized by comprising a reagent A and a reagent B, wherein the mass ratio of the reagent A to the reagent B is 0.25-4, and in the staining agent,
the reagent A comprises:
0.6-1.2 g of neutral red dye; the neutral red dye is a weakly basic dye, is positively charged under near neutral and alkaline conditions, and interacts with phosphate groups on chromatin molecules containing negatively charged groups in cells, carboxyl groups on protein molecules and sialic acid molecules on cell membranes to show red;
100g of dye solvent; the dye solvent is one or more of purified water and an organic solvent, and the organic solvent can be one or a combination of methanol, ethanol, glycol, amyl alcohol, glycerol and dimethyl sulfoxide;
the reagent B comprises:
10 mM-600 mM of buffering agent; the buffer is one or a mixture of more of 2- (N-morpholine) ethanesulfonic acid, 1, 3-di [ (trihydroxymethyl) methylamino ] propane, piperazine-1, 4-diethylsulfonic acid, 3- (N-morpholinyl) propanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, trimethylolaminomethane-hydrochloric acid, barbituric acid-sodium hydrochloride, propionic acid-sodium propionate and acetic acid-sodium acetate;
0.1-8 g of cell membrane damaging agent, wherein the cell membrane damaging agent is: CH (CH)3(CH2)n1COOR，n1A combination of 1, 2 or 3,wherein R is Na, H or K;
0-4 g of complexing agent; the complexing agent is citric acid, citrate or ethylenediamine tetraacetate;
0.01-10 g of pH regulator; the pH regulator is acetic acid, propionic acid or hydrochloric acid;
100g of solvent.
2. A use method of a staining agent capable of quickly developing color after staining cells in urine is characterized by comprising the following steps:
step S1, preparing a reagent A and a reagent B, wherein the reagent A comprises:
0.6-1.2 g of neutral red dye; the neutral red dye is a weakly basic dye, is positively charged under near neutral and alkaline conditions, and interacts with phosphate groups on chromatin molecules containing negatively charged groups in cells, carboxyl groups on protein molecules and sialic acid molecules on cell membranes to show red;
100g of dye solvent; the dye solvent is one or more of purified water and an organic solvent, and the organic solvent can be one or a combination of methanol, ethanol, glycol, amyl alcohol, glycerol and dimethyl sulfoxide;
the reagent B comprises:
10 mM-600 mM of buffering agent; the buffer is one or a mixture of more of 2- (N-morpholine) ethanesulfonic acid, 1, 3-di [ (trihydroxymethyl) methylamino ] propane, piperazine-1, 4-diethylsulfonic acid, 3- (N-morpholinyl) propanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, trimethylolaminomethane-hydrochloric acid, barbituric acid-sodium hydrochloride, propionic acid-sodium propionate and acetic acid-sodium acetate;
0.1-8 g of cell membrane damaging agent, wherein the cell membrane damaging agent is: CH (CH)3(CH2)n1COOR，n11, 2 or 3, wherein R is Na, H or K;
0-4 g of complexing agent; the complexing agent is citric acid, citrate or ethylenediamine tetraacetate;
0.01-10 g of pH regulator; the pH regulator is acetic acid, propionic acid or hydrochloric acid;
100g of a solvent;
step S2, mixing the prepared reagent A and the prepared reagent B according to the mass ratio of 0.25-4 to obtain a coloring agent, and then dropwise adding the coloring agent to the concentrated or non-concentrated urine sample;
and step S3, dyeing for 5-30S to obtain a dyed urine sample.
3. The use method of the staining agent for rapid color development after staining the cells in the urine according to claim 2, wherein the preparation method of the reagent A and the preparation method of the reagent B are respectively as follows:
preparation of reagent A:
a1, adding a dye solvent and a chemical dye into a reagent bottle in sequence to obtain a first mixed solution A1;
a2, placing a reagent bottle containing the first mixed solution A1 on a shaking table to be fully dissolved for 2-5 hours to obtain a second mixed solution A2;
A. filtering the second mixed solution A2, marking the filtrate as a reagent A, and storing for later use;
preparation of reagent B:
b1, adding a buffering agent, a complexing agent and a cell membrane damaging agent into purified water, and fully dissolving the materials to obtain a first mixed solution B1;
b2, adding a pH regulator into the first mixed solution B1 to adjust the pH value to 4.8-7.0, and obtaining a second mixed solution B2;
B. the second mixed solution B2 was filtered, and the filtrate was labeled as reagent B and kept for future use.
4. The use method of the staining agent capable of rapidly developing color after staining the cells in the urine according to claim 2, wherein the mass ratio of the reagent A to the reagent B in the staining agent is 0.67-1.5.
5. The use method of the staining agent for rapid color development after staining the cells in the urine according to claim 2, wherein the volume ratio of the urine sample to the staining agent is 1-8.
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| CN109481141B (en) * | 2019-01-16 | 2021-01-26 | 青岛大学附属医院 | Method for staining anterior lens capsule in over-mature cataract surgery |
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| CN114459982B (en) * | 2022-01-17 | 2024-05-31 | 桂林优利特医疗电子有限公司 | Urine component analysis method based on flow cytometry principle |
| CN114441272A (en) * | 2022-01-27 | 2022-05-06 | 北京艾迪康医学检验实验室有限公司 | Exfoliative cell staining solution and preparation method thereof |
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