CN110132899A - A kind of hemolytic agent for leukocyte differential count - Google Patents
A kind of hemolytic agent for leukocyte differential count Download PDFInfo
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- CN110132899A CN110132899A CN201810105118.9A CN201810105118A CN110132899A CN 110132899 A CN110132899 A CN 110132899A CN 201810105118 A CN201810105118 A CN 201810105118A CN 110132899 A CN110132899 A CN 110132899A
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- agent
- buffer
- concentration
- hemolytic agent
- differential count
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- 239000003219 hemolytic agent Substances 0.000 title claims abstract description 61
- 210000000265 leukocyte Anatomy 0.000 title claims abstract description 32
- 239000003093 cationic surfactant Substances 0.000 claims abstract description 24
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 239000000872 buffer Substances 0.000 claims abstract description 21
- 239000004094 surface-active agent Substances 0.000 claims abstract description 17
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 12
- 230000003204 osmotic effect Effects 0.000 claims abstract description 11
- 230000003196 chaotropic effect Effects 0.000 claims abstract description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- 125000000623 heterocyclic group Chemical group 0.000 claims description 18
- 125000002091 cationic group Chemical group 0.000 claims description 12
- 125000000217 alkyl group Chemical group 0.000 claims description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 9
- 125000003342 alkenyl group Chemical group 0.000 claims description 9
- 125000005211 alkyl trimethyl ammonium group Chemical group 0.000 claims description 9
- 239000002736 nonionic surfactant Substances 0.000 claims description 9
- 125000000304 alkynyl group Chemical group 0.000 claims description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 6
- 150000002367 halogens Chemical class 0.000 claims description 6
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical group O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 5
- YWFWDNVOPHGWMX-UHFFFAOYSA-N n,n-dimethyldodecan-1-amine Chemical compound CCCCCCCCCCCCN(C)C YWFWDNVOPHGWMX-UHFFFAOYSA-N 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- CXRFDZFCGOPDTD-UHFFFAOYSA-M Cetrimide Chemical compound [Br-].CCCCCCCCCCCCCC[N+](C)(C)C CXRFDZFCGOPDTD-UHFFFAOYSA-M 0.000 claims description 3
- XGEGHDBEHXKFPX-UHFFFAOYSA-N N-methylthiourea Natural products CNC(N)=O XGEGHDBEHXKFPX-UHFFFAOYSA-N 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Chemical class OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- GCKVRHFRDJAIGL-UHFFFAOYSA-M [NH4+].[Br-].C(CCCCCCCCCCC)[P+](CC)(C)C.[Br-] Chemical compound [NH4+].[Br-].C(CCCCCCCCCCC)[P+](CC)(C)C.[Br-] GCKVRHFRDJAIGL-UHFFFAOYSA-M 0.000 claims description 3
- PTWAIEVJHCYRSB-UHFFFAOYSA-M [NH4+].[Br-].C(CCCCCCCCCCCCCCCCC)[P+](CC)(C)C.[Br-] Chemical compound [NH4+].[Br-].C(CCCCCCCCCCCCCCCCC)[P+](CC)(C)C.[Br-] PTWAIEVJHCYRSB-UHFFFAOYSA-M 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 150000001299 aldehydes Chemical class 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 3
- BGKUZGVLFHGANI-UHFFFAOYSA-M dodecyl-ethyl-dimethylazanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)CC BGKUZGVLFHGANI-UHFFFAOYSA-M 0.000 claims description 3
- XWBDWHCCBGMXKG-UHFFFAOYSA-N ethanamine;hydron;chloride Chemical compound Cl.CCN XWBDWHCCBGMXKG-UHFFFAOYSA-N 0.000 claims description 3
- KSCHLNBLIAOANF-UHFFFAOYSA-M ethyl-hexadecyl-dimethylazanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)CC KSCHLNBLIAOANF-UHFFFAOYSA-M 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 150000002460 imidazoles Chemical class 0.000 claims description 3
- 125000002883 imidazolyl group Chemical group 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 3
- XGEGHDBEHXKFPX-NJFSPNSNSA-N methylurea Chemical compound [14CH3]NC(N)=O XGEGHDBEHXKFPX-NJFSPNSNSA-N 0.000 claims description 3
- 229960005181 morphine Drugs 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- GJWJYZYRBDJBHK-UHFFFAOYSA-N quinoline;sodium Chemical compound [Na].N1=CC=CC2=CC=CC=C21 GJWJYZYRBDJBHK-UHFFFAOYSA-N 0.000 claims description 3
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 claims description 3
- 150000003839 salts Chemical class 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
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 2
- HCBIBCJNVBAKAB-UHFFFAOYSA-N Procaine hydrochloride Chemical compound Cl.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 HCBIBCJNVBAKAB-UHFFFAOYSA-N 0.000 claims description 2
- VBIIFPGSPJYLRR-UHFFFAOYSA-M Stearyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)C VBIIFPGSPJYLRR-UHFFFAOYSA-M 0.000 claims description 2
- SWLVFNYSXGMGBS-UHFFFAOYSA-N ammonium bromide Chemical compound [NH4+].[Br-] SWLVFNYSXGMGBS-UHFFFAOYSA-N 0.000 claims description 2
- 239000007979 citrate buffer Substances 0.000 claims description 2
- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 claims description 2
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 claims description 2
- RXHDXDIEHWVFOC-UHFFFAOYSA-M ethyl-dimethyl-octadecylazanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC RXHDXDIEHWVFOC-UHFFFAOYSA-M 0.000 claims description 2
- CQWWONKCVFKXRE-UHFFFAOYSA-N n,n-dimethylethanamine;hydrobromide Chemical compound [Br-].CC[NH+](C)C CQWWONKCVFKXRE-UHFFFAOYSA-N 0.000 claims description 2
- 229960001309 procaine hydrochloride Drugs 0.000 claims description 2
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- CEYYIKYYFSTQRU-UHFFFAOYSA-M trimethyl(tetradecyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC[N+](C)(C)C CEYYIKYYFSTQRU-UHFFFAOYSA-M 0.000 claims description 2
- BGHCVCJVXZWKCC-UHFFFAOYSA-N tetradecane Chemical compound CCCCCCCCCCCCCC BGHCVCJVXZWKCC-UHFFFAOYSA-N 0.000 claims 2
- 150000001345 alkine derivatives Chemical class 0.000 claims 1
- AJXBTRZGLDTSST-UHFFFAOYSA-N amino 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)ON AJXBTRZGLDTSST-UHFFFAOYSA-N 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 claims 1
- 230000031709 bromination Effects 0.000 claims 1
- 238000005893 bromination reaction Methods 0.000 claims 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 210000000601 blood cell Anatomy 0.000 abstract description 15
- 210000004027 cell Anatomy 0.000 abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 239000000975 dye Substances 0.000 abstract description 6
- 210000004698 lymphocyte Anatomy 0.000 abstract description 5
- 210000001616 monocyte Anatomy 0.000 abstract description 5
- 238000000790 scattering method Methods 0.000 abstract description 5
- 210000004493 neutrocyte Anatomy 0.000 abstract description 4
- 238000012827 research and development Methods 0.000 abstract description 4
- 238000004043 dyeing Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 22
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002949 hemolytic effect Effects 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- -1 primary amine salt Chemical class 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 3
- 239000000980 acid dye Substances 0.000 description 3
- 210000003979 eosinophil Anatomy 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- AUBSNUSTVUZGCC-UHFFFAOYSA-N [NH4+].[Br-].C(C)[PH3+].[Br-] Chemical compound [NH4+].[Br-].C(C)[PH3+].[Br-] AUBSNUSTVUZGCC-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 2
- HXWGXXDEYMNGCT-UHFFFAOYSA-M decyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCC[N+](C)(C)C HXWGXXDEYMNGCT-UHFFFAOYSA-M 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- YFVKHKCZBSGZPE-UHFFFAOYSA-N 1-(1,3-benzodioxol-5-yl)-2-(propylamino)propan-1-one Chemical compound CCCNC(C)C(=O)C1=CC=C2OCOC2=C1 YFVKHKCZBSGZPE-UHFFFAOYSA-N 0.000 description 1
- MPNXSZJPSVBLHP-UHFFFAOYSA-N 2-chloro-n-phenylpyridine-3-carboxamide Chemical compound ClC1=NC=CC=C1C(=O)NC1=CC=CC=C1 MPNXSZJPSVBLHP-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 244000248349 Citrus limon Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 241001208007 Procas Species 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000009304 pastoral farming Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 150000003866 tertiary ammonium salts Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- AISMNBXOJRHCIA-UHFFFAOYSA-N trimethylazanium;bromide Chemical compound Br.CN(C)C AISMNBXOJRHCIA-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (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 discloses a kind of hemolytic agents for leukocyte differential count, including following several components: cationic surfactant, nonionic surface active agent, buffer, osmotic pressure regulator, chaotropic agent and the antibacterial agent that pH is 3~7.Hemolytic agent of the invention can make leucocyte be divided into four lymphocyte, monocyte, neutrophil leucocyte, acidophic cell subgroups on the blood cell analyzer that Supreme Being steps.Hemolytic agent of the present invention is free of dyestuff, can be classified by laser scattering method to leucocyte to cell without dyeing, reduce the harm to environment, the research and development cost and complexity of reagent and instrument are reduced, suitable for vast medical institutions.
Description
Technical field
The present invention relates to blood cell analysis detection field more particularly to a kind of haemolysis for leukocyte differential count
Agent.
Background technique
Normal peripheral white blood cells are generally divided into five classes, i.e. lymphocyte (Lym), monocyte (Mon), neutral grain is thin
Born of the same parents (Neu), eosinophil (Eos), basophilic granulocyte (Baso).In clinical analysis field, for different types of white
Cell is accurately detected for diagnosing and research has very important effect.
Leucocyte can only be divided into cellule, centre using impedance method by three classification blood cell analyzers currently on the market
Three cell, maxicell subgroups, cannot accurately classify to leucocyte, in clinical analysis field, there are certain limitations
Property.With the continuous improvement of today's society scientific and technological level, five classification blood cell analyzers generally use laser scattering method or group
It closes other methods to classify to leucocyte, so that testing result is more accurate.
And use laser scattering method to leukocyte differential count firstly the need of using the surfactant in hemolytic agent to leucocyte
Punching processing is carried out, different degrees of shrinkage can occur after handling via hemolytic agent for different types of leucocyte；Then logical
It crosses nucleic acid dye and dye marker, the scattered light intensity of the different leucocytes after nucleic acid staining dye is carried out to nucleus
It is different.Dyestuff using maximum the disadvantage is that be more toxic, not only endanger the health of operator, can also be to environment
It pollutes.Meanwhile nucleic acid dye is at high cost, increases the complexity of reagent and instrument research and development.
The method that leukocyte differentiation is four classes is used in United States Patent (USP) 518733 and contains quaternary ammonium salt cationic surface-active
The hemolytic agent of agent makes the leucocyte of four kinds of subgroups that different degrees of shrinkage occur.The nucleic acid dye in reagent is to leucocyte simultaneously
Core is dyed, so that the grazing shot luminous intensity of the leucocyte of four kinds of subgroups generates difference.Pass through point of the scattering light of different angle
Four points of groups to leucocyte are realized in analysis.Although the patent realizes relatively accurately distinguishes leucocyte, but its reagent is ground
The complexity for sending out cost and instrument increases, and cost performance is not suitable for those middle-size and small-size clients.
A large amount of long-chain quaternary ammonium surfactant has been used in hemolytic agent disclosed in CN 200810106893.2, kind
Class is more, also very big to leukocytoclastic while to erythrocyte hemolysis.
Leucocyte can be divided by hemolytic agent disclosed in United States Patent (USP) 4751179 on its corresponding cellanalyzer
Four kinds of subgroups, however the reagent needs to maintain when acting on 70 DEG C or so of temperature with haemocyte, could occur well anti-
It answers.
Therefore, the existing technology needs to be improved and developed.
Summary of the invention
The purpose of the present invention is to provide a kind of new hemolytic agents for leukocyte differential count, can be by leukocyte differentiation
Four kinds of lymphocyte (Lym), monocyte (Mon), neutrophil leucocyte (Neu) and eosinophil (Eos) subgroups, do not need
Any dyestuff is added, the complexity of reagent and instrument research and development is reduced, it is intended to which the leukocyte differential count technology in the prior art that solves exists
Problem complicated for operation.
Technical scheme is as follows:
A kind of hemolytic agent for leukocyte differential count, wherein including following components:
Cationic surfactant, nonionic surface active agent, the buffer that pH is 2~9, osmotic pressure regulator, rush
Solvent and antibacterial agent；
The cationic surfactant includes at least one quaternary cationics and at least one heterocycle
Cationoid surfactant；
The concentration of the quaternary cationics is 0.1~100g/L；
The concentration of the heterocyclic cationic surfactant is 0.1~100g/L；
The concentration of the nonionic surfactant is preferably 1~50mL/L；
The pH of the buffer is maintained in the range of 2~9；
The concentration of the osmotic pressure regulator is 1~20g/L；
The concentration of the chaotropic agent is 1~20mL/L；
The concentration of the antibacterial agent is 0.01~10g/L or 0.01~10mL/L.
The hemolytic agent for leukocyte differential count, wherein
The quaternary cationics have the structure as shown in formula (I):
In formula (I), R1 is selected from alkyl, alkenyl or the alkynyl of C8-20；R2, R3, R4 be selected from the alkyl of C1-4, alkenyl or
Person's alkynyl；X- is selected from halogen anionoid or oxygen-containing acid ion；
The heterocyclic cationic surfactant has the structure as shown in formula (II):
In formula (II), R1 is alkyl, alkenyl or the alkynyl of C8-22；X- is selected from halogen anionoid.
The hemolytic agent for leukocyte differential count, wherein the quaternary cationics are selected from eight alkane
Base trimethylammonium bromide, eight alkyl trimethyl ammonium chlorides, ten alkyl trimethyl ammonium bromides, decyl trimethyl ammonium chloride, 12
Alkyl trimethyl ammonium bromide, dodecyl trimethyl ammonium chloride, dodecyl dimethyl ethyl phosphonium bromide ammonium, dodecyl dimethyl
Ethyl ammonium chloride, tetradecyltrimethylammonium bromide, tetradecyl trimethyl ammonium chloride, dodecyldimethylamine base ethyl phosphonium bromide
Ammonium, dodecyldimethylamine base ethyl ammonium chloride, cetyl trimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecane
Base dimethyl ethyl ammonium bromide, cetyl dimethylethyl ammonium chloride, Cetyltrimethylammonium bromide, octadecyl front three
One or more of ammonium chloride, octadecyldimethyl ethyl phosphonium bromide ammonium, octadecyldimethyl ethyl ammonium chloride are appointed
Meaning combination；
The heterocyclic cationic surfactant is in morphine ring, pyridine ring, imidazole ring, pyridine ring and quinoline ring
It is one or more of.
The hemolytic agent for leukocyte differential count, wherein the heterocyclic cationic surfactant is pyridine ring
Cationoid surfactant.
The hemolytic agent for leukocyte differential count, wherein the nonionic surfactant be selected from polyoxyethylene-type,
One or more of ethylene glycol, TWEEN Series, Qula flow-through surfactant.
The hemolytic agent for leukocyte differential count, wherein the buffer is selected from PBS buffer solution, Acetic acid-sodium acetate
One of buffer, citrate buffer, Tris buffer, borate buffer, BR buffer or carbonic acid buffer are several
Kind.
The hemolytic agent for leukocyte differential count, wherein the osmotic pressure regulator is selected from methylurea or inorganic salts.
The hemolytic agent for leukocyte differential count, wherein the chaotropic agent is selected from ethyl alcohol, ethylene glycol, propyl alcohol, the third two
One or more of alcohol, glycerine, isopropanol, butanol and butanediol any combination.
The hemolytic agent for leukocyte differential count, wherein the antibacterial agent in the hemolytic agent is selected from hydrochloric acid Proca
One of cause, the western quinoline sodium of piperazine, Prolin series, imidazoles series, NaF, phenol, aldehydes, amide series are a variety of any
Combination.
The hemolytic agent for leukocyte differential count, wherein the concentration of the quaternary cationics is
0.1~75g/L；
The concentration of the heterocyclic cationic surfactant is 0.2~60g/L；
The concentration of the nonionic surfactant is 1~15mL/L；
The pH of the buffer is 3~7；
The concentration of the chaotropic agent is 3~15mL/L；
The concentration of the antibacterial agent is 0.01~5g/L or 0.01~5mL/L.
Beneficial effects of the present invention: can be made on the blood cell analyzer that Supreme Being steps using hemolytic agent of the invention white thin
Born of the same parents are divided into four lymphocyte, monocyte, neutrophil leucocyte, acidophic cell subgroups.Hemolytic agent of the present invention is free of
Dyestuff can be classified to cell to leucocyte by laser scattering method without dyeing, reduce the harm to environment, reduce
The research and development cost and complexity of reagent and instrument, suitable for vast medical institutions.
Detailed description of the invention
Fig. 1 a- Fig. 1 d is the test result figure for carrying out hemolytic experiment in the present invention using the hemolytic agent that embodiment 1 is configured.
Fig. 2 a- Fig. 2 d is the test result figure for carrying out hemolytic experiment in the present invention using the hemolytic agent that embodiment 2 is configured.
Fig. 3 a- Fig. 3 d is the test result figure for carrying out hemolytic experiment in the present invention using the hemolytic agent that embodiment 3 is configured.
Fig. 4 a- Fig. 4 d is the test result figure for carrying out hemolytic experiment in the present invention using the hemolytic agent that embodiment 4 is configured.
Fig. 5 a- Fig. 5 d is respectively the eosino-lympho-mononutro-gram of embodiment 1-5 in the present invention.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer and more explicit, right as follows in conjunction with drawings and embodiments
The present invention is further described.
The present invention provides a kind of hemolytic agents for leukocyte differential count, including following several components:
Cationic surfactant, nonionic surface active agent, the buffer that pH is 3~7, osmotic pressure regulator, rush
Solvent and antibacterial agent.
The cationic surfactant is selected from primary amine salt, secondary amine salt, tertiary ammonium salt, quaternary ammonium salt, heterocycle, salt cation table
A kind of or a few class in the activating agent of face.Preferably, the cationic surfactant includes at least one quaternary ammonium salt cationic table
Face activating agent and at least one heterocyclic cationic surfactant.
The effect of the quaternary cationics is lysed erythrocyte, eliminates red blood cell to leukocyte differential count
It influences, while dialogue cell membrane being wanted to be handled, so that lymph, monokaryon, neutrophil leucocyte, acidophic cell occur in various degree
Shrinkage.
The quaternary cationics have the structure as shown in formula (I):
Wherein, R1 can be selected from alkyl, alkenyl or the alkynyl of C8-20；R2, R3, R4 can be independently selected from C1-4's
Alkyl, alkenyl or alkynyl.X- is selected from halogen anionoid or oxygen-containing acid ion.
The quaternary cationics be selected from eight alkyl trimethyl ammonium bromides, eight alkyl trimethyl ammonium chlorides,
Ten alkyl trimethyl ammonium bromides, decyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide, trimethyl chlorination
Ammonium, dodecyl dimethyl ethyl phosphonium bromide ammonium, dodecyl dimethyl ethyl ammonium chloride, tetradecyltrimethylammonium bromide, ten
Tetraalkyl trimethyl ammonium chloride, dodecyldimethylamine base ethyl phosphonium bromide ammonium, dodecyldimethylamine base ethyl ammonium chloride, cetyl
Trimethylammonium bromide, hexadecyltrimethylammonium chloride, cetyldimethylethylambromide bromide ammonium, cetyl dimethylethyl
Ammonium chloride, Cetyltrimethylammonium bromide, octadecyltrimethylammonium chloride, octadecyldimethyl ethyl phosphonium bromide ammonium, ten
Any combination of one or more of eight alkyl dimethyl ethyl ammonium chlorides.The concentration of the quaternary surfactant is
0.1~100g/L, preferably 0.1~75g/L.
Heterocyclic cationic surfactant dosage in the hemolytic agent is compared with quaternary cationics dosage
Lack, effect mainly can do faint processing after quaternary surfactant lysed erythrocyte to leucocyte, make it
Show different degrees of contraction.
The heterocyclic cationic surfactant has the structure as shown in formula (II):
Wherein, R1 is alkyl, alkenyl or the alkynyl of C8-22；X- is selected from halogen anionoid.
The heterocyclic cationic surfactant can be selected from morphine ring, pyridine ring, imidazole ring, pyridine ring and quinoline ring
One or more of.Heterocyclic cationic surfactant in the hemolytic agent is preferably that pyridine ring cationoid surface is living
Property agent.The concentration of the heterocyclic cationic surfactant is 0.1~100g/L, preferably 0.2~60g/L.
The nonionic surfactant also has certain haematolysis ability to red blood cell, while dialogue cell membrane also will do it
Certain processing.Nonionic surfactant there is also certain emulsification, enables surfactant in hemolytic agent
It is evenly dispersed.The nonionic surfactant is selected from polyoxyethylene-type, ethylene glycol, TWEEN Series, Qula flow-through surface-active
One or more of agent.The concentration of the nonionic surfactant is preferably 1~50mL/L, preferably 1~15mL/L.
It is constant that buffer in the hemolytic agent is able to maintain pH, keeps a metastable physical and chemical ring for blood cell
Border, convenient for the precise classification and detection of leucocyte.The buffer is selected from PBS buffer solution, Acetic acid-sodium acetate buffer, lemon
One or more of phthalate buffer, Tris buffer, borate buffer, BR buffer or carbonic acid buffer.The buffering
In the range of the pH of liquid maintains 2~9, preferably 3~7 range.
The hemolytic agent will make leucocyte keep certain form, and osmotic pressure is also an important factor.The haemolysis
Osmotic pressure regulator in agent is selected from methylurea or inorganic salts.The concentration of the osmotic pressure regulator is 1~20g/L.
There are the organic matters such as a large amount of Surfactant in the hemolytic agent, in order to promote it preferably to dissolve, Ke Yishi
When the stronger substance of addition polarity promote its dissolution in aqueous solution.The chaotropic agent be selected from ethyl alcohol, ethylene glycol, propyl alcohol,
One or more of propylene glycol, glycerine, isopropanol, butanol and butanediol any combination.The concentration of the chaotropic agent is 1
~20mL/L, preferably 3~15mL/L.
The effect of antibacterial agent in the hemolytic agent is prevented molten to effectively kill bacterium and inhibit the growth of microorganism
Blood agent is gone mouldy.Antibacterial agent in the hemolytic agent is selected from procaine hydrochloride, the western quinoline sodium of piperazine, Prolin series, imidazoles
One of series, NaF, phenol, aldehydes, amide series or a variety of any combination.The concentration of the antibacterial agent be 0.01~
10g/L or 0.01~10mL/L, preferably 0.01~5g/L or 0.01~5mL/L.
Above-mentioned raw materials directly are required to be uniformly mixed by the preparation method of hemolytic agent of the present invention according to concentration, and accurate
Constant volume is filtered, and is used for the specific blood cell analyzer of our company.The application method of the hemolytic agent and market are routinely molten
Blood agent is identical, and this will not be repeated here.
Further detailed description is done to the present invention below by specific embodiment.
Embodiment 1
25 parts of clinical vein whole blood samples are taken, steps in Supreme Being and is configured on DF50 blood cell analyzer using the present embodiment 1
Hemolytic agent tested, while being tested on stepping auspicious BC-5180 blood cell analyzer using stepping auspicious original-pack hemolytic agent,
The two result is handled in Excel, to step the test result on auspicious BC-5180 as ordinate, is surveyed using the present embodiment 1
Test result is abscissa, as shown in Fig. 1 a- Fig. 1 d, it can be seen from the figure that result and stepping the test result on auspicious BC-5180
With preferable linear relationship.
Embodiment 2
25 parts of clinical vein whole blood samples are taken, steps in Supreme Being and is configured on DF50 blood cell analyzer using the present embodiment 2
Hemolytic agent tested, while carrying out (using auspicious original-pack hemolytic agent is stepped) on stepping auspicious BC-5180 blood cell analyzer real
It tests, the two result is handled in Excel, to step the test result on auspicious BC-5180 as ordinate, using the present embodiment
2 test results are abscissa.As shown in Fig. 2 a- Fig. 2 d, it can be seen from the figure that result and stepping the test on auspicious BC-5180
As a result there is preferable linear relationship.
Embodiment 3
25 parts of clinical vein whole blood samples are taken, steps in Supreme Being and is configured on DF50 blood cell analyzer using the present embodiment 3
Hemolytic agent tested, while carrying out (using auspicious original-pack hemolytic agent is stepped) on stepping auspicious BC-5180 blood cell analyzer real
It tests, the two result is handled in Excel, to step the test result on auspicious BC-5180 as ordinate, using the present embodiment
3 test results are abscissa.As shown in Fig. 3 a- Fig. 3 d, it can be seen from the figure that result and stepping the test on auspicious BC-5180
As a result there is preferable linear relationship.
Embodiment 4
It is 6.0 with the hemolytic agent pH that this component and proportion are completed
25 parts of clinical vein whole blood samples are taken, steps in Supreme Being and is configured on DF50 blood cell analyzer using the present embodiment 4
Hemolytic agent tested, while being tested on stepping auspicious BC-5180 blood cell analyzer using stepping auspicious original-pack hemolytic agent,
The two result is handled in Excel, to step the test result on auspicious BC-5180 as ordinate, is surveyed using the present embodiment 4
Test result is abscissa.As shown in Figure 4 a- shown in Figure 4 d, it can be seen from the figure that result and stepping the test result on auspicious BC-5180
With preferable linear relationship.
(A), (B), (C) in Fig. 5 a- Fig. 5 d, that (D) respectively corresponds embodiment 1, embodiment 2, embodiment 3, embodiment 4 is molten
Blood agent corresponding eosino-lympho-mononutro-gram when being tested on DF50.Leucocyte is acted in the channel DIFF class and this hemolytic agent, by this
After the hemolytic agent processing of invention, four kinds of subgroups are divided by laser scattering method, are lymphocyte, monocyte, neutrality respectively
Granulocyte, eosinophil.Fig. 5 a (A) is the eosino-lympho-mononutro-gram of 1 test result of embodiment, it can be seen that four kinds of classification are thin
The scatter plot of born of the same parents, it is well dispersed between every kind of leucocyte scatterplot；Fig. 5 b (B) is the eosino-lympho-mononutro-gram of 2 test result of embodiment,
Four kinds of leucocytes can be clearly separated, and the scatterplot coincidence of each section is smaller, and classifying quality is preferable；Fig. 5 c (C) is the survey of embodiment 3
The eosino-lympho-mononutro-gram of test result, four kinds of leucocytes can be clearly separated, and the extent of polymerization of each section is preferable, classifying quality
Preferably；Fig. 5 d (D) is the eosino-lympho-mononutro-gram of 4 result of embodiment, and four kinds of leucocyte results can be clearly separated, each section
Extent of polymerization it is preferable, classifying quality is obvious.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention
Protect range.
Claims (10)
1. a kind of hemolytic agent for leukocyte differential count, which is characterized in that including following components:
Cationic surfactant, nonionic surface active agent, the buffer that pH is 2~9, osmotic pressure regulator, chaotropic agent
And antibacterial agent；
The cationic surfactant includes at least one quaternary cationics and at least one heterocyclic sun
Ionic surface active agent；
The concentration of the quaternary cationics is 0.1~100g/L；
The concentration of the heterocyclic cationic surfactant is 0.1~100g/L；
The concentration of the nonionic surfactant is preferably 1~50mL/L；
The pH of the buffer is maintained in the range of 2~9；
The concentration of the osmotic pressure regulator is 1~20g/L；
The concentration of the chaotropic agent is 1~20mL/L；
The concentration of the antibacterial agent is 0.01~10g/L or 0.01~10mL/L.
2. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that
The quaternary cationics have the structure as shown in formula (I):
In formula (I), R1 is selected from alkyl, alkenyl or the alkynyl of C8-20；R2, R3, R4 are selected from alkyl, alkenyl or the alkynes of C1-4
Base；X- is selected from halogen anionoid or oxygen-containing acid ion；
The heterocyclic cationic surfactant has the structure as shown in formula (II):
In formula (II), R1 is alkyl, alkenyl or the alkynyl of C8-22；X- is selected from halogen anionoid.
3. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the quaternary ammonium salt cationic table
Face activating agent is selected from eight alkyl trimethyl ammonium bromides, eight alkyl trimethyl ammonium chlorides, ten alkyl trimethyl ammonium bromides, ten alkyl three
Ammonio methacrylate, dodecyl trimethyl ammonium bromide, dodecyl trimethyl ammonium chloride, dodecyl dimethyl ethyl phosphonium bromide
Ammonium, dodecyl dimethyl ethyl ammonium chloride, tetradecyltrimethylammonium bromide, tetradecyl trimethyl ammonium chloride, the tetradecane
Base dimethyl ethyl ammonium bromide, dodecyldimethylamine base ethyl ammonium chloride, cetyl trimethylammonium bromide, cetyl front three
Ammonium chloride, cetyldimethylethylambromide bromide ammonium, cetyl dimethylethyl ammonium chloride, octadecyl trimethyl bromination
Ammonium, octadecyltrimethylammonium chloride, octadecyldimethyl ethyl phosphonium bromide ammonium, in octadecyldimethyl ethyl ammonium chloride
One or more of any combination；
The heterocyclic cationic surfactant is selected from one of morphine ring, pyridine ring, imidazole ring, pyridine ring and quinoline ring
Or it is several.
4. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the heterocyclic cation form
Face activating agent is pyridine ring cationoid surfactant.
5. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the non-ionic surface active
Agent is selected from one or more of polyoxyethylene-type, ethylene glycol, TWEEN Series, Qula flow-through surfactant.
6. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the buffer is selected from PBS
Buffer, Acetic acid-sodium acetate buffer, citrate buffer, Tris buffer, borate buffer, BR buffer or carbonic acid
One or more of buffer.
7. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the osmotic pressure regulator choosing
From methylurea or inorganic salts.
8. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the chaotropic agent is selected from second
One or more of alcohol, ethylene glycol, propyl alcohol, propylene glycol, glycerine, isopropanol, butanol and butanediol any combination.
9. the hemolytic agent according to claim 1 for leukocyte differential count, which is characterized in that the antibacterial in the hemolytic agent
Agent is in procaine hydrochloride, the western quinoline sodium of piperazine, Prolin series, imidazoles series, NaF, phenol, aldehydes, amide series
One or more any combination.
10. any hemolytic agent for leukocyte differential count according to claim 1~9, which is characterized in that the quaternary ammonium salt
The concentration of cationic surfactant is 0.1~75g/L；
The concentration of the heterocyclic cationic surfactant is 0.2~60g/L；
The concentration of the nonionic surfactant is 1~15mL/L；
The pH of the buffer is 3~7；
The concentration of the chaotropic agent is 3~15mL/L；
The concentration of the antibacterial agent is 0.01~5g/L or 0.01~5mL/L.
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