CN107167356A - In a kind of urine after cell dyeing quick colour-developing coloring agent and its application method - Google Patents
In a kind of urine after cell dyeing quick colour-developing coloring agent and its application method Download PDFInfo
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- CN107167356A CN107167356A CN201710207744.4A CN201710207744A CN107167356A CN 107167356 A CN107167356 A CN 107167356A CN 201710207744 A CN201710207744 A CN 201710207744A CN 107167356 A CN107167356 A CN 107167356A
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- reagent
- coloring agent
- urine
- agent
- cell
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- 210000002700 urine Anatomy 0.000 title claims abstract description 94
- 238000004043 dyeing Methods 0.000 title claims abstract description 73
- 239000003086 colorant Substances 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 132
- 210000004027 cell Anatomy 0.000 claims abstract description 54
- 239000002904 solvent Substances 0.000 claims abstract description 21
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 19
- 230000006378 damage Effects 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
- 239000008139 complexing agent Substances 0.000 claims abstract description 12
- 239000003002 pH adjusting agent Substances 0.000 claims abstract description 8
- 230000007935 neutral effect Effects 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims description 56
- 239000000975 dye Substances 0.000 claims description 42
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 22
- 238000002360 preparation method Methods 0.000 claims description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 10
- 239000003960 organic solvent Substances 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 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 7
- 239000008213 purified water Substances 0.000 claims description 7
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 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
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 229910052700 potassium Inorganic materials 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- PDIDTMXYILXCLW-UHFFFAOYSA-M sodium;propanoate;propanoic acid Chemical compound [H+].[Na+].CCC([O-])=O.CCC([O-])=O PDIDTMXYILXCLW-UHFFFAOYSA-M 0.000 claims description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 3
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 claims description 3
- 229960002319 barbital Drugs 0.000 claims description 3
- -1 disodium hydrogen Chemical class 0.000 claims description 3
- 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 3
- LJSOLTRJEQZSHV-UHFFFAOYSA-L potassium;sodium;hydron;hydroxide;phosphate Chemical compound [OH-].[Na+].[K+].OP(O)([O-])=O LJSOLTRJEQZSHV-UHFFFAOYSA-L 0.000 claims description 3
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 3
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 3
- 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 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 229940009662 edetate Drugs 0.000 claims description 2
- KOGINMITGZVQBD-UHFFFAOYSA-N ethanesulfonic acid;piperazine Chemical compound CCS(O)(=O)=O.C1CNCCN1 KOGINMITGZVQBD-UHFFFAOYSA-N 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 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 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- SMWDFEZZVXVKRB-UHFFFAOYSA-N anhydrous quinoline Natural products N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims 1
- 239000006172 buffering agent Substances 0.000 claims 1
- 210000003743 erythrocyte Anatomy 0.000 abstract description 18
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000010186 staining Methods 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 5
- 230000007170 pathology Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 238000004040 coloring Methods 0.000 description 11
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 238000001914 filtration Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 108010077544 Chromatin Proteins 0.000 description 7
- 210000003483 chromatin Anatomy 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000005034 decoration Methods 0.000 description 6
- 210000004940 nucleus Anatomy 0.000 description 6
- KVYRCBOUKXJXDK-UHFFFAOYSA-N 3,4-dimethylphenazine-1,2-diamine hydrochloride Chemical class Cl.C1=CC=CC2=NC3=C(C)C(C)=C(N)C(N)=C3N=C21 KVYRCBOUKXJXDK-UHFFFAOYSA-N 0.000 description 5
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 5
- 230000003139 buffering effect Effects 0.000 description 5
- 210000000805 cytoplasm Anatomy 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 5
- 235000019260 propionic acid Nutrition 0.000 description 5
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical class [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 5
- 235000010334 sodium propionate Nutrition 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- 241000235342 Saccharomycetes Species 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 241001502500 Trichomonadida Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 3
- 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 description 3
- 239000001294 propane Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000736199 Paeonia Species 0.000 description 2
- 235000006484 Paeonia officinalis Nutrition 0.000 description 2
- 229920000297 Rayon Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000015165 citric acid Nutrition 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- AEDZKIACDBYJLQ-UHFFFAOYSA-N ethane-1,2-diol;hydrate Chemical compound O.OCCO AEDZKIACDBYJLQ-UHFFFAOYSA-N 0.000 description 2
- OXYHAZHGSRTVAP-UHFFFAOYSA-N ethane-1,2-diol;methanol;hydrate Chemical compound O.OC.OCCO OXYHAZHGSRTVAP-UHFFFAOYSA-N 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000010399 physical interaction Effects 0.000 description 2
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108010017480 Hemosiderin Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- SDNHZJXLRQQDIJ-UHFFFAOYSA-N O.OCC(O)CO.C(CO)O Chemical compound O.OCC(O)CO.C(CO)O SDNHZJXLRQQDIJ-UHFFFAOYSA-N 0.000 description 1
- MJTSOZOWJWOKAY-UHFFFAOYSA-L P(=O)([O-])([O-])O.[Na+].[Na+].P Chemical compound P(=O)([O-])([O-])O.[Na+].[Na+].P MJTSOZOWJWOKAY-UHFFFAOYSA-L 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 238000011882 arthroplasty Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- NGPZWOOKVNRELB-UHFFFAOYSA-N ethane-1,2-diol;methanol Chemical compound OC.OCCO NGPZWOOKVNRELB-UHFFFAOYSA-N 0.000 description 1
- YMBNBZFZTXCWDV-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2,3-triol Chemical compound OCCO.OCC(O)CO YMBNBZFZTXCWDV-UHFFFAOYSA-N 0.000 description 1
- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002652 macrocyte Anatomy 0.000 description 1
- 230000008384 membrane barrier Effects 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
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- LBFCRPJWCIHPSX-UHFFFAOYSA-N pentan-1-ol;hydrate Chemical compound O.CCCCCO LBFCRPJWCIHPSX-UHFFFAOYSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 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
- 238000000746 purification Methods 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- 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
Abstract
The present invention provides the coloring agent and its application method of quick colour-developing after cell dyeing in a kind of urine, wherein, including reagent A and reagent B, and reagent A and reagent B mass ratio are 0.25 ~ 4, in coloring agent, reagent A includes:Neutral 0.6 ~ 1.2g of red；Dye solvent 100g；Reagent B includes:Buffer 10mM ~ 600mM；0.1 ~ 8g of cell membrane damage agent；0 ~ 4g of complexing agent；0.01 ~ 10g of pH adjusting agent；Solvent 100g.The coloring agent is not required to pre-process cell in urine just can be to its direct staining, and developing time is short, and simple to operate, detection efficiency is high；In addition, the coloring agent does not influence the form of the red blood cell with pathology sense, the red blood cell sample of high concentration will not be caused to assemble, be conducive to improving the accuracy of testing result, it is possible to achieve automatic detection.
Description
Technical field
The present invention relates to the coloring agent of quick colour-developing after cell dyeing in in-vitro diagnosis field, more particularly to a kind of urine and
Its application method.
Background technology
Visible component in urine includes the Multiple components such as red blood cell, leucocyte, cast, epithelial cell.Visible component point
Analyse significant to urinary system, the detection for closing on reproductive system and other systemic diseases.Numerous studies show, urine
In crystallization, saccharomycete can disturb the analysis of urine erythrocyte, substantial amounts of viscose silk and mycelia can cause the vacation sun that cast is counted, in vain
Cell and the unconspicuous leucocyte of microphage and nucleus and macrocyte, leucocyte and trichomonad, spore and red blood cell
Form is close, and hyalina and sperm are colourless, and the probability for flase drop missing inspection occur is larger, therefore chemical staining is to pathology sense
The discriminating of particle is significant.Although the colouring method of existing formation in urine is more, equal Shortcomings, such as
Sternheimer decoration methods, Sternheimer-Malbin decoration methods, Burhe-Muhlberg decoration methods and A Li Xinlan-in
Property red colouring method, not only dyeing time is long（>1 minute）, allogenic cell colour developing heterogeneity, and Color is by the pH of urine specimen
Value limitation, in addition, composite dye dyeing easily disturbance caused thing occurs；May-Giemsa, Rui Shi, Ji Shi, auspicious lucky complex staining
The sample preparation technology of the fixed decoration method such as method is complicated, is unfavorable for the purpose for improving the speed examined and realizing Automated inspection；
The special stainings such as oil red decoration method, iodine staining, Goodpasture's staining, hemosiderin decoration method are only applicable to urine
The analysis of a certain class visible component in liquid sample.
Therefore, prior art has yet to be improved and developed.
The content of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide quickly show after cell dyeing in a kind of urine
The coloring agent and its application method of color, it is intended to when solving complicated existing urine cell dyeing method sample preparation technology, dyeing
Between long, red cell morphology influenceed by dyeing liquor and easily occur aggregation and the problem of be unfavorable for realizing automatic detection.
Technical scheme is as follows：
In a kind of urine after cell dyeing quick colour-developing coloring agent, wherein, including reagent A and reagent B, and reagent A and reagent B
The mass ratio of consumption is 0.25 ~ 4, in coloring agent,
Reagent A includes:
Neutral 0.6 ~ 1.2g of red；
Dye solvent 100g；
Reagent B includes:
Buffer 10mM ~ 600mM；
0.1 ~ 8g of cell membrane damage agent；
0 ~ 4g of complexing agent；
0.01 ~ 10g of pH adjusting agent；
Solvent 100g.
In described urine after cell dyeing quick colour-developing coloring agent, wherein, the dye solvent is purified water and to have
One or more in machine solvent.
In described urine after cell dyeing quick colour-developing coloring agent, wherein, the organic solvent can be methanol, second
One kind or its composition in alcohol, ethylene glycol, amylalcohol, glycerine, dimethyl sulfoxide (DMSO).
In described urine after cell dyeing quick colour-developing coloring agent, wherein, the buffer be 2-（N- morpholines）
Ethyl sulfonic acid, 1,3- bis- [（Trihydroxy methyl）Methylamino] propane, the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, 3-（N- morpholinyls）Propane sulfonic acid, 3-
（N- morpholinyls）- 2- hydroxy-propanesulfonic acids, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-phosphorus
Acid dihydride potassium, potassium dihydrogen phosphate-sodium hydroxide, trishydroxymethylaminomethane-hydrochloric acid, barbital sodium-hydrochloric acid, propionic acid-sodium propionate,
One group or several groups of mixture in acetic acid-sodium acetate.
In described urine after cell dyeing quick colour-developing coloring agent, wherein, the cell membrane damage agent is shown under being
One or more in machine compound：
CH3(CH2)n1COOR, n1=0,1,2 or 3, wherein R are Na, H or K；
In described urine after cell dyeing quick colour-developing coloring agent, wherein, the complexing agent be citric acid, citrate or
Edetate.
In a kind of urine after cell dyeing the coloring agent of quick colour-developing application method, wherein, including：
Step S1, reagent preparation A and reagent B；
Step S2, the reagent A and reagent B of preparation be first mixed to get coloring agent for 0.25 ~ 4 ratio in mass ratio, then
The coloring agent is added drop-wise to after concentration or in the urine specimen of non-concentrated；
After step S3,5 ~ 30s of dyeing, the urine specimen after being dyed.
In described urine after cell dyeing the coloring agent of quick colour-developing application method, wherein, reagent A and reagent B's
Preparation method is respectively：
The preparation of reagent A：
A1, solution diluent, dye solvent and chemical dye be added in reagent bottle according to the sequence, obtain the first mixing molten
Liquid A1；
A2, by the above-mentioned reagent bottle containing the first mixed solution A 1 be placed on shaking table fully dissolving 2 ~ 5 hours, obtain the second mixing
Solution A 2；
A, second mixed solution A 2 is filtered, filtrate is labeled as reagent A, and save backup；
Reagent B preparation：
B1, by buffer, complexing agent and cell membrane damage agent add purified water in, it is fully dissolved and obtain the first mixed solution
B1；
B2, into the first mixed solution B1 add pH adjusting agent adjust pH value to 4.8-7.0, obtain the second mixed solution
B2；
B, the second mixed solution B2 is filtered, filtrate is labeled as reagent B, and save backup.
In described urine after cell dyeing the coloring agent of quick colour-developing application method, wherein, the coloring agent pilot scale
Agent A and reagent B mass ratio are 0.25 ~ 4.
In described urine after cell dyeing the coloring agent of quick colour-developing application method, wherein, the coloring agent with urine
The volume ratio of liquid sample is 1 ~ 8.
Beneficial effect：In the urine that the present invention is provided after cell dyeing quick colour-developing reagent, be not required to cell in urine
Being pre-processed just can be to its direct staining, and developing time is short, and simple to operate, detection efficiency is high；In addition, the coloring agent not shadow
The form of the red blood cell with pathology sense is rung, the red blood cell sample of high concentration will not be caused to assemble, is conducive to improving and examines
Survey the accuracy of result and realize the purpose of automatic detection.
Brief description of the drawings
Fig. 1,2,12 are that the coloring agent and colouring method prepared according to embodiment in the present invention 4 enters to the leucocyte in urine
The design sketch of row dyeing.
Fig. 3,4,5 are the coloring agent and colouring method prepared according to embodiment in the present invention 1 to the epithelial cell in urine
（Fig. 4 containing clues cell）The design sketch dyed.
Fig. 6,7,8 are that the coloring agent and colouring method prepared according to embodiment in the present invention 2 is carried out to the cast in urine
The design sketch of dyeing.
Fig. 9,10,11 are the coloring agent and colouring method prepared according to embodiment in the present invention 3 to the red blood cell in urine
The design sketch dyed.
Embodiment
The present invention provides the coloring agent and its application method of quick colour-developing after cell dyeing in a kind of urine, to make the present invention
Purpose, technical scheme and effect it is clearer, clear and definite, the present invention is described in more detail below.It should be appreciated that this place
The specific embodiment of description only to explain the present invention, is not intended to limit the present invention.
In the urine that the present invention is provided after cell dyeing quick colour-developing coloring agent, wherein, including reagent A and reagent B, and
Reagent A and reagent B mass ratio are 0.25 ~ 4, in coloring agent,
Reagent A includes:
Neutral 0.6 ~ 1.2g of red；
Dye solvent 100g；
Reagent B includes:
Buffer 10mM ~ 600mM；
0.1 ~ 8g of cell membrane damage agent；
0 ~ 4g of complexing agent；
0.01 ~ 10g of pH adjusting agent；
Solvent 100g.
Wherein, chemical dye selection can be diffused into inside cell membrane, and the organic dyestuff that nucleus cytoplasm is distinguished.
The mode that chemical dye interacts with cell is divided into Physical interaction and chemical interaction, and Physical interaction follows phase
Like compatibility principle, chemical interaction includes electrostatic interaction, interaction of hydrogen bond, Van der Waals force, hydrophobic force and covalent bond,
Wherein electrostatic interaction is most commonly seen, is also one kind that maximum is contributed in motive power.The diffusion velocity of dyestuff is by dyestuff
Molecular weight effects, molecular weight is bigger, and diffusion velocity is slower.
Based on above-mentioned action principle, in the present invention, the preferred neutral red of chemical dye, as a kind of alkalescent dyestuff,
Phosphate group on its positively charged under weakly acidic pH and alkalescence condition, with the chromatin molecule of the intracellular group containing negative electricity, albumen
The aobvious red of sialic acid molecule interaction on carboxyl and cell membrane on matter molecule.Chromatin is fine and close in nucleus, color depth,
Chromatin and albumen distribution are loose in cytoplasm, colour shallow.It can be clearlyed distinguish according to the cellular morphology after dyeing various thin
Born of the same parents' type.In addition, the dyestuff is applied widely, can be many to the leucocyte in urine, epithelial cell, red blood cell, sperm etc.
Composition is planted to be dyed.In the present invention, mass fraction of the neutral red in A and B mixed liquors is 0.12% ~ 0.96%, properly
The neutral red of consumption can not only be carried out cell in the cell after rapid dyeing, and dyeing, nucleus and cytoplasm
Clear in structure, is easily differentiated, and is conducive to improving checkability.
Solvent of the present invention is the one or more in purified water and organic solvent, and its effect is to enable chemical dye long-term
It is stable to preserve, and the coloring agent of preparation is kept preferable Color.Wherein, purified water is conducive to increasing the dissolving of salt
Degree, while can be diluted to organic solvent, prevents organic solvent from excessively causing the RBC deformation in urine specimen.This
Outside, the determination method of purifying water consumption need to meet two aspect requirements：One is ensureing that red blood cell is not deformed upon, secondly being to ensure that
Dye liquor is without precipitate.In the present invention, consumption mass fraction of the reagent A in A and B mixed liquors is 20% ~ 80%.The dye solvent
Organic solvent can be methanol, ethanol, ethylene glycol, amylalcohol, glycerine, dimethyl sulfoxide (DMSO)（DMSO）Or its composition.As methanol-
Ethanol, ethylene glycol-methanol, DMSO- ethylene glycol, ethylene glycol-glycerine or a variety of organic solvents mixing compounding.Organic solvent and water
Between can also be compounded after as dye solvent, such as methanol-water, alcohol-water, ethylene glycol-water, amylalcohol-water, glycerine-
Water, dimethyl sulfoxide (DMSO)（DMSO）- water, ethylene glycol-methanol-water, ethylene glycol-alcohol-water, ethylene glycol-glycerol-water, glycerine-
Methanol-water, preferably, ethylene glycol-water or ethylene glycol-methanol-water are preferable as the effect of dye solvent.
The main function of buffer is regulation urine ph values and osmotic pressure, maintains red cell morphology, while ensureing relatively permanent
Fixed dyeing circumstances, make dyestuff close to the Color of the cell in different urine specimens.For urine system, selection buffering
The buffering range of agent is advisable in pH=4.8-7.0, and the buffering range of preferred reducing is in pH=4.8-6.8, preferably, selection buffering
The buffering range of agent is in pH=5.0-6.2.2- can be used in buffer（N- morpholines）Ethyl sulfonic acid（MES）, 1,3- bis- [（Three（Hydroxyl first
Base）Methylamino] propane（Bis-tris）, piperazine-the ethyl sulfonic acid of Isosorbide-5-Nitrae-two（PIPES）、3-（N- morpholinyls）Propane sulfonic acid（MOPS）、3-
（N- morpholinyls）- 2- hydroxy-propanesulfonic acids（MOPSO）In one kind；Can also for 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, barbital sodium-hydrochloric acid,
One group therein of propionic acid-sodium propionate, acetic acid-sodium acetate, can also be the composite buffer system between above-mentioned buffer.As selected
1,3- bis- [（Three（Methylol）Methylamino] propane（Bis-tris）With the compound system of propionic acid-sodium propionate, can be directly by urine
PH value and osmotic pressure be adjusted to appropriate value, be easy to the progress of follow-up dyeing course.
Buffer play pH value adjustment effect when, in reagent A and the mixed solution of reagent B, concentration be 10mmol/L ~
600mmol/L is more suitable；To ensure preferably Color, buffer is dense in reagent A and the mixed solution of reagent B
Spend for 10mmol/L ~ 200mmol/L it is more suitable.
Cell membrane damage material be it is a kind of can destroy the organic compound molecule of living cell membrane barrier, it is mainly made
For phospholipid bilayer, i.e. the hydrophobic side of itself is embedded in into phospholipid bilayer, make phospholipid bilayer arrangement loose, this
Sample, the phospholipid bilayer of surface area increase is easy to small molecule to be diffused into the inside of cell, such as dye molecule, and it can be from impaired
Cell membrane is freely spread to cell interior, and nucleus and cytoplasm are dyed.Simultaneously, it is desirable to which cell membrane damage material is being acted on
During phospholipid bilayer, it is impossible to influence cellular morphology, the shape of the red blood cell with diagnostic significance of fragility can not be especially influenceed
State.The consumption of cell membrane damage material to observe 5 minutes under the microscope, and red blood cell is without obvious deformation, and red blood cell count(RBC) is constant
It is defined.The cell membrane damage material can be following three class, be respectively：
CH3(CH2)n1COOR, wherein, n1=0,1,2 or 3, R can be Na, H or K；It is preferred that, when n1=1 or 2, cell membrane
The effect of damaging agents preferably, and works as cell membrane damage material in reagent A and the mixed solution of reagent B, mass percent
For 0.1% ~ 8% when, during preferably 1-4%, cellular damage material can ensure with diagnostic significance cellular morphology it is impregnable
Under the premise of, the cross-film speed of dye molecule is improved, rapid dyeing is carried out to the cell in urine.
Complexing agent energy and alkali metal, rare earth element and transition metal etc. form stable water soluble complex, and it is mainly made
Be reduce urine in interference of the amorphism salt to dyeing course.In the present invention, complexing agent can be citrate or second
Ethylenediamine tetraacetic acid (EDTA)（EDTA）Salt, the consumption of complexing agent is that in reagent A and the mixed solution of reagent B, mass concentration is with 0-4%
It is advisable.
PH value regulator, for the pH value of further adjustment coloring agent, it is ensured that the pH environment of dyeing, coloring agent can be fast
Speed is dyed to cell in urine, and its form will not be impacted.It is preferred that, pH adjusting agent can be acetic acid, propionic acid,
Hydrochloric acid etc., 4.8-7.0 is adjusted to by reagent B pH value.
Solvent B effect is fully to dissolve the salt of addition, obtains the reagent B of stable homogeneous, is easy to coordinate with reagent A
Using, and then the cell in urine is dyed.In the present invention, the solvent in reagent B is preferably purified water, not only to adding
Different material have preferable dissolubility, and the cell in urine will not be impacted.
In addition, the present invention also provides a kind of application method of the coloring agent of quick colour-developing after cell dyeing in urine, including：
Step S1, reagent preparation A and reagent B；
Step S2, the mentioned reagent A and reagent B of preparation be first mixed to get coloring agent for 0.25 ~ 4 ratio in mass ratio, then
Above-mentioned coloring agent is added drop-wise in the urine specimen after concentration；
After step S3,5 ~ 30s of dyeing, the urine specimen after being dyed.
Preferably, the step of reagent preparation A includes：Dye solvent is first added into reagent bottle, chemical dye is added, obtained
To the first mixed solution A 1, the above-mentioned reagent bottle containing the first mixed solution A 1 is placed on shaking table fully dissolving 2 ~ 5 hours, obtained
To the second mixed solution A 2, above-mentioned second mixed solution A 2 is filtered, filtrate reagent A is labeled as, and save backup.System
The step of standby reagent B, includes：Buffer, complexing agent and cell membrane damage agent are added into solvent purification water, fully after dissolving
To the first mixed solution B1, above-mentioned first mixed solution B1 pH value is then adjusted to 4.8-7.0 with pH adjusting agent, second is obtained
Mixed solution B2, is finally filtered to above-mentioned second mixed solution B2, filtrate is labeled as into reagent B, and save backup.
The mentioned reagent A prepared and reagent B are mixed to get coloring agent for 0.25 ~ 4 ratio in mass ratio, then will be upper
State in the urine specimen that coloring agent is added drop-wise to after non-concentrated or concentration.It is preferred that, reagent A and reagent B mass ratio for 0.67 ~
1.5, when reagent A and reagent B are mixed according to aforementioned proportion, dyeing agent solution is more homogeneous, and property is more stablized, dyeing speed
Degree is faster.And coloring agent and the volume ratio that urine specimen is added are 1 ~ 8, because, when the volume ratio of urine and coloring agent exists
When in above range, coloring agent fast and effeciently can be dyed the cell in urine, and the cell in urine will not be by
The coloring agent of addition destroys original structure and form, is conducive to improving the accuracy of testing result.
Added into the urine after above-mentioned concentration after appropriate coloring agent, dye 5 ~ 30s, the urine sample after being dyed
This.In addition, the method dyeing liquor in the present invention, is applicable not only to sediments microscope inspection, while may apply to streamed digital image
In analytical technology, be conducive to improving the accuracy of detection efficiency and testing result.
Embodiment 1
1st, the preparation of coloring agent
Reagent A:
5.00g methanol, 95.00g ethylene glycol and 1.20g dimethyl diaminophenazine chlorides are added according to sequencing in brown reagent bottle, the is obtained
One mixed solution A 1；Afterwards, the brown reagent bottle that will be equipped with above-mentioned first mixed solution A 1 is placed on shaking table, and middling speed dissolving 3 is small
When so that medicine is completely dissolved, and obtains the second mixed solution A 2；Then above-mentioned second mixed solution A 2 is entered with 0.22um filter membranes
Row filtering, reagent A is labeled as by the solution after filtering, is placed in preserving in brown bottle.
Reagent B:
100.00g purified waters, 4.20g are added in reagent bottle（200mM）1,3- bis- [（Three（Methylol）Methylamino] propane
（Bis-tris）, 8.00g sodium propionates, fully dissolving after obtain the first mixed solution B；Added into above-mentioned first mixed solution B1
Propionic acid, makes the pH value of solution B 1 in the range of 5.50 ± 0.05（25±1℃）；Then mixed with 0.22 μm of filter membrane to above-mentioned second
Solution B 2 is filtered, and the solution after filtering is labeled as into reagent B, is placed in preserving in reagent bottle.
2nd, colouring method
By mentioned reagent A and reagent B with 1:1 mass ratio is mixed, and is shaken up and is configured to use dyeing liquor.Urine specimen can pass through
The urine specimen of concentration, is first placed in tally, is then directly added drop-wise to the dyeing liquor prepared by the method concentration of centrifugation
The ratio of concentration or non-concentrated urine specimen, dye liquor and urine specimen is 1：Urine after being dyed after 8, dyeing 10s
Sample.
Embodiment 2
1st, the preparation of coloring agent
Reagent A:
100.00g ethylene glycol and 1.20g dimethyl diaminophenazine chlorides are added according to sequencing in brown reagent bottle, the first mixed solution is obtained
A1；Afterwards, the brown reagent bottle that will be equipped with above-mentioned first mixed solution A 1 is placed on shaking table, and middling speed dissolves 3 hours so that medicine
It is completely dissolved, obtains the second mixed solution A 2；Then above-mentioned second mixed solution A 2 is filtered with 0.22um filter membranes, incited somebody to action
Solution after filter is labeled as reagent A, is placed in preserving in brown bottle.
Reagent B:
100.00g purified waters, 2.10g are added in reagent bottle（50mM）2-（N- morpholines）Ethyl sulfonic acid monohydrate（MES）、
4.00g sodium propionates, 4.00g EDTA-K2Fully the first mixed solution B1 is obtained after dissolving；With propionic acid by the first mixed solution B1
PH value adjust in the range of 6.00 ± 0.05（25±1℃）；Then above-mentioned second mixed solution B2 is entered with 0.22 μm of filter membrane
Row filtering, reagent B is labeled as by the solution after filtering, is placed in preserving in reagent bottle.
2nd, colouring method
By mentioned reagent A and reagent B with 0.25：1 mass ratio is mixed, and dyeing liquor i.e. is configured to after shaking up.Will be logical
The urine specimen for crossing centrifugal process concentration is placed in tally, and the dyeing liquor prepared is directly then added drop-wise into concentration or non-concentrated
In urine specimen, the volume ratio of dye liquor and urine specimen is 1：Urine specimen after being dyed after 1, dyeing 30s.
Embodiment 3
1st, the preparation of coloring agent
Reagent A:
100g glycerine and 0.6g dimethyl diaminophenazine chlorides are added according to sequencing in brown reagent bottle, the first mixed solution A is obtained；It
Afterwards, the brown reagent bottle that will be equipped with above-mentioned first mixed solution A 1 is placed on shaking table, and middling speed dissolves 3.5 hours so that medicine is complete
Fully dissolved, obtains the second mixed solution A 2；Then above-mentioned second mixed solution A 2 is filtered with 0.22um filter membranes, will filtered
Solution afterwards is labeled as reagent A, is placed in preserving in brown bottle.
Reagent B:
100g purified waters, 0.61g are added in reagent bottle（50mM）Trishydroxymethylaminomethane（Tris）, 8g sodium propionates and
The first mixed solution B1 is obtained after 1.9g citric acids, fully dissolving；With propionic acid by the first mixed solution B1 pH value adjust to
In the range of 6.70 ± 0.05（25±1℃）；Then above-mentioned second mixed solution B2 is filtered with 0.22 μm of filter membrane, will filtered
Solution afterwards is labeled as reagent B, is placed in preserving in reagent bottle.
2nd, colouring method
By mentioned reagent A and reagent B with 4：1 mass ratio is mixed, and dyeing liquor i.e. is configured to after shaking up.It will pass through
The urine specimen of centrifugal process concentration is placed in tally, and the dyeing liquor prepared is directly then added drop-wise into concentration or non-concentrated urine
In liquid sample, the volume ratio of dye liquor and urine specimen is 3：Urine specimen after being dyed after 7, dyeing 5s.
Embodiment 4
1st, the preparation of coloring agent
Reagent A:
5g methanol, 95g ethylene glycol and 0.8 dimethyl diaminophenazine chloride are added according to sequencing in brown reagent bottle, the first mixing is obtained molten
Liquid A1；Afterwards, the brown reagent bottle that will be equipped with above-mentioned first mixed solution A 1 is placed on shaking table, and middling speed dissolves 4 hours so that medicine
Product are completely dissolved, and obtain the second mixed solution A 2；Then above-mentioned second mixed solution A 2 is filtered with 0.22um filter membranes, will
Solution after filtering is labeled as reagent A, is placed in preserving in brown bottle.
Reagent B:
100g purified waters, 4g Bis-tris, 8g sodium propionates are added in reagent bottle, fully the first mixed solution is obtained after dissolving
B1；The first mixed solution B1 pH value is adjusted in the range of 5.00 ± 0.05 using propionic acid（25±1℃）；Then with 0.22 μm
Filter membrane is filtered to above-mentioned second mixed solution B2, and the solution after filtering is labeled as into reagent B, is placed in protecting in reagent bottle
Deposit.
2nd, colouring method
By mentioned reagent A and reagent B with 3：2 mass ratio is mixed, and dyeing liquor i.e. is configured to after shaking up.It will pass through
The urine specimen of centrifugal process concentration is placed in tally, and the dyeing liquor prepared is directly then added drop-wise into concentration or non-concentrated urine
In liquid sample, the volume ratio of dye liquor and urine specimen is 1：Urine specimen after being dyed after 6, dyeing 20s.
Embodiment 5
1st, the preparation of coloring agent
Reagent A:
5g methanol, 95g ethylene glycol and 1.2g dimethyl diaminophenazine chlorides are added according to sequencing in brown reagent bottle, afterwards, be will be equipped with
The brown reagent bottle for stating the first mixed solution A 1 is placed on shaking table, and middling speed is dissolved 5 hours so that medicine is completely dissolved, and obtains the
Two mixed solution As 2；Then above-mentioned second mixed solution A 2 is filtered with 0.22um filter membranes, the solution after filtering is marked
For reagent A, it is placed in preserving in brown bottle.
Reagent B:
100g purified waters, 3g Bis-tris, 6g sodium propionates are added in reagent bottle, fully the first mixed solution is obtained after dissolving
B1；With acetic acid by the range of the first mixed solution B1 pH value regulation 5.80 ± 0.05（25±1℃）；Then with 0.22 μm of filter membrane
Above-mentioned second mixed solution B2 is filtered, the solution after filtering is labeled as reagent B, is placed in preserving in reagent bottle.
2nd, colouring method
By mentioned reagent A and reagent B with 2：3 mass ratio is mixed, and dyeing liquor i.e. is configured to after shaking up.It will pass through
The urine specimen of centrifugal process concentration is placed in tally, and the dyeing liquor prepared is directly then added drop-wise into concentration or non-concentrated urine
In liquid sample, the volume ratio of dye liquor and urine specimen is 1：Urine specimen after being dyed after 4, dyeing 15s.
In the present invention, leukocyte cell core is can be seen that because chromatin is intensive from Fig. 1 and Fig. 2, peony, cell is dyed
Nuclear boundary is clear, and cytoplasm is relatively fewer because of chromatin and protein ingredient, dyes light.
As can be seen that epithelial cells core is intensive because of chromatin in Fig. 3,4,5, peony is dyed, nuclear boundary is clear
Clear, cytoplasm is relatively fewer because of chromatin and protein ingredient, dyes light.
As can be seen that the dyeing of cast surface glycoprotein is light in Fig. 6 and Fig. 7, inclusion is deep because containing protein ingredient, dyeing,
It is clear and legible.
As can be seen that the light dye of trichomonad, dyestuff adds the contrast of sample and background, so visible trichomonad flagellum in Fig. 8
And vesicular nucleus, easily differentiate with the leucocyte after dyeing；
It is high-visible after core and the tail dyeing of sperm as can be seen that dyestuff adds the contrast of sample and background in Fig. 9,
Easily differentiate with achromophil saccharomycete.
As can be seen that the red blood cell in urine is not dyed or light dye in Figure 10 and Figure 11, red blood cell is clear-cut.
Figure 12 can be seen that viscose silk and saccharomycete are not dyed, but dyeing background is because of the effect of dyestuff, slightly color, photo
Contrast is deepened so that achromophil saccharomycete easily differentiates with leucocyte.
It is believed that in the urine of the present invention after cell dyeing the coloring agent and its application method of quick colour-developing for urine
In different cells, be respectively provided with preferable Color.
In summary, the invention provides the reagent of quick colour-developing after cell dyeing in a kind of urine and its application method,
The coloring agent can carry out rapid dyeing to the cell in urine, and the form of red blood cell is not damaged, and application method is simple, dyeing
Cell colour developing afterwards is homogeneous, can not only improve efficiency and the degree of accuracy of cervical arthroplasty, flow cytometric-digital color imaging technique
Development significance it is far-reaching.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can
To be improved or converted according to the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention
Protect scope.
Claims (10)
1. in a kind of urine after cell dyeing quick colour-developing coloring agent, it is characterised in that including reagent A and reagent B, and reagent
A and reagent B mass ratio are 0.25 ~ 4, in coloring agent,
Reagent A includes:
Neutral 0.6 ~ 1.2g of red；
Dye solvent 100g；
Reagent B includes:
Buffer 10mM ~ 600mM；
0.1 ~ 8g of cell membrane damage agent；
0 ~ 4g of complexing agent；
0.01 ~ 10g of pH adjusting agent；
Solvent 100g.
2. in urine according to claim 1 after cell dyeing quick colour-developing coloring agent, it is characterised in that the dyestuff
Solvent is the one or more in purified water and organic solvent.
3. in urine according to claim 2 after cell dyeing quick colour-developing coloring agent, it is characterised in that it is described organic
Solvent can be one kind or its composition in methanol, ethanol, ethylene glycol, amylalcohol, glycerine, dimethyl sulfoxide (DMSO).
4. in urine according to claim 1 after cell dyeing quick colour-developing coloring agent, it is characterised in that the buffering
Agent is 2-（N- morpholines）Ethyl sulfonic acid, 1,3- bis- [（Trihydroxy methyl）Methylamino] propane, the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, 3-（N-
Quinoline base）Propane sulfonic acid, 3-（N- morpholinyls）- 2- hydroxy-propanesulfonic acids, disodium hydrogen phosphate-citric acid, disodium hydrogen phosphate-biphosphate
Sodium, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, trishydroxymethylaminomethane-hydrochloric acid, barbital sodium-
One group or several groups of mixture in hydrochloric acid, propionic acid-sodium propionate, acetic acid-sodium acetate.
5. in urine according to claim 1 after cell dyeing quick colour-developing coloring agent, it is characterised in that the cell
Membrane damage agent is：
CH3(CH2)n1COOR, n1=0,1,2 or 3, wherein R are Na, H or K.
6. in urine according to claim 1 after cell dyeing quick colour-developing coloring agent, it is characterised in that the complexing
Agent is citric acid, citrate or edetate.
7. in a kind of urine after cell dyeing the coloring agent of quick colour-developing application method, it is characterised in that including：
Step S1, reagent preparation A and reagent B；
Step S2, the reagent A and reagent B of preparation be first mixed to get coloring agent for 0.25 ~ 4 ratio in mass ratio, then
The coloring agent is added drop-wise in the urine specimen after concentration or non-concentrated；
After step S3,5 ~ 30s of dyeing, the urine specimen after being dyed.
8. in urine according to claim 7 after cell dyeing the coloring agent of quick colour-developing application method, its feature exists
In reagent A and reagent B preparation method are respectively：
The preparation of reagent A：
A1, solution diluent, dye solvent and chemical dye be added in reagent bottle according to the sequence, obtain the first mixing molten
Liquid A1；
A2, by the above-mentioned reagent bottle containing the first mixed solution A 1 be placed on shaking table fully dissolving 2 ~ 5 hours, obtain the second mixing
Solution A 2；
A, second mixed solution A 2 is filtered, filtrate is labeled as reagent A, and save backup；
Reagent B preparation：
B1, by buffer, complexing agent and cell membrane damage agent add purified water in, it is fully dissolved and obtain the first mixed solution
B1；
B2, into the first mixed solution B1 add pH adjusting agent adjust pH value to 4.8 to 7.0, obtain the second mixed solution
B2；
B, the second mixed solution B2 is filtered, filtrate is labeled as reagent B, and save backup.
9. in urine according to claim 7 after cell dyeing the coloring agent of quick colour-developing application method, its feature exists
In reagent A and reagent B quality ratio scope are 0.67 ~ 1.5 in the coloring agent.
10. in urine according to claim 7 after cell dyeing the coloring agent of quick colour-developing application method, its feature exists
In the volume ratio of the urine specimen and coloring agent is 1 ~ 8.
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| CN109612807A (en) * | 2018-12-29 | 2019-04-12 | 湖北伽诺美生物科技有限公司 | A kind of urinary formed element dyeing liquor |
| CN112213172A (en) * | 2020-09-16 | 2021-01-12 | 迪瑞医疗科技股份有限公司 | Stable vaginal secretion visible component staining solution and preparation method thereof |
| CN112362435A (en) * | 2020-11-05 | 2021-02-12 | 深圳安侣医学科技有限公司 | Cell staining reagent and cell staining method |
| 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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| 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 |
| CN112213172A (en) * | 2020-09-16 | 2021-01-12 | 迪瑞医疗科技股份有限公司 | Stable vaginal secretion visible component staining solution and preparation method thereof |
| CN112362435A (en) * | 2020-11-05 | 2021-02-12 | 深圳安侣医学科技有限公司 | Cell staining reagent and cell staining method |
| CN114459982A (en) * | 2022-01-17 | 2022-05-10 | 桂林优利特医疗电子有限公司 | Urine visible 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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