CN103120153A - Exfoliative cells preserving fluid - Google Patents
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- CN103120153A CN103120153A CN2012104881485A CN201210488148A CN103120153A CN 103120153 A CN103120153 A CN 103120153A CN 2012104881485 A CN2012104881485 A CN 2012104881485A CN 201210488148 A CN201210488148 A CN 201210488148A CN 103120153 A CN103120153 A CN 103120153A
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Abstract
The invention discloses an exfoliative cells preserving fluid which is prepared from a pH buffering agent, an osmotic pressure maintenance agent, preservatives, a fixing agent for maintaining cellular morphology, an anticoagulant, a mucus softener, an antimicrobial reagent, a cleaning agent, a humectant and red blood cell destroying components. The components in the preserving fluid are reasonable in proportioning, and the exfoliative cells can be preserved at a long time under normal temperature, wherein the longest time can reach 2 years; mucus can be sufficiently dissolved and the red cells can be partially destroyed; a film production effect is good, cellular distribution is very even, cellular morphology is perfect, cytoplasm and cell nucleus demarcation is distinct, gradation is clear, and cytoplasm and cell nucleus transparency is very good, and the exfoliative cells preserving fluid can be simultaneously used for the HPV-DNA (human papillomavirus-deoxyribonucleic acid), Chlamydia and immunohistochemical test; the special liquid base preserving fluid used for the cytologic examination of other parts can be provided, an imported product can be completely replaced, and the cellular constituent with diagnostic significance can be sufficiently preserved; and the exfoliative cells preserving fluid is low in configuration cost and easy to popularize.
Description
Technical field
The present invention relates to a kind of exfoliated cell preservative fluid.
Background technology
The human body living cells has physiological function, biologic activity and intrinsic cellular morphology and structure, has diagnostic value aspect a lot.If place but it is broken away from original living environment, the physiological function of cell, biologic activity and intrinsic cellular morphology and structure will change, conventional method is exactly that the cell that comes off is carried out freezing preservation, and freezing preservation needs special freezing equipment, more expensive, and, due to the harm that exists in freezing, course of defrosting cellular damage, people and cause cytometaplasia or distortion affects follow-up work.
As the screening method of cervical lesions, what relate in traditional Pap smear process draws materials, is coated with the factors such as tablet quality, cell separation and all can seriously reduce the positive rate of cell.The cytology expert has released a kind of sectioning cells diagnostic method-ThinPrep liquid-based cytology test technology (Thinprep Cytology Test; Be called for short TCT), it mainly loses non-diagnosis impurity by technical finesse, makes cheap observation thin-layer cell sheet clearly, makes to read the sheet person more easily observes, and diagnostic accuracy obviously improves.
ThinPrep liquid-based cytology test technology (TCT) has changed the method for operating of Pap smear in the past, sample is put into cell-preservation liquid after taking out immediately, avoided in conventional Pap smear process, the cell that is retained on sampler abandons phenomenon with the cell transition drying with sampler.Cell in preservation liquid is through procedure treatment, thin smear is made in random sampling, undesired cell in thin slice is easily observed, and moist cell nucleus structure is more clear, be convenient to differentiate, can detect more rudimentary pathology and some more serious pathologies, be popular new technology in the world at present.Adopt the ThinPrep liquid-based cytology test technology to improve 13% to paracytic rate of correct diagnosis, the recall rate of the above pathology of scaly epithelium has been improved 65%, have advantages of that traditional smear is incomparable.In the TCT technology, the most key reagent is cell-preservation liquid.At present, on market 40%～50% alcohols commonly used as preserving liquid, but alcohol concentration is greater than the 50% fixing high protein of mobility, but protein just forms sediments immediately, can not well transfer to slide, this brings puzzlement to cytolgical examination, can cause simultaneously the cell surface distortion.If can not fix for a long time preservation less than 20% cell, easily cause cell degradation.
The TCT technology has become one of best recommend method of screening cervical carcinoma, for the early diagnosis and therapy of cervical carcinoma provides very clear and definite diagnosis basis, especially in today of modern technologies high speed development, apply widely and seem particularly urgent and important, therefore develop suitable cervical exfoliated cell preservative fluid extremely urgent.Thus, the solution that cast-off cells are preserved has appearred on market being specifically designed to, Bai Shi as new in the U.S. (
Cytology Test) the preservation liquid (PresetverCyt Slution) of producing, this preservations liquid is and the matching used reagent of its machine Thinpreper-2000, but clinical expensive, and the defective that exists haemocyte processing and cell nucleus to dwindle.for reducing application cost, better preserve and process cell, domestic researcher has developed a kind of new preservation formula of liquid, it as application reference number is 200610017591.9 the disclosed a kind of exfoliated cell preservative fluid (publication number: CNl834233A) of Chinese patent application, the preservation liquid of this application consists of the following composition, by volume part is counted: 40 parts～60 parts of methyl alcohol or ethanol, 20 parts～40 parts of phosphate buffer or trishydroxymethylaminomethane hydrochloride buffers, 5 parts～10 parts, formaldehyde or acetone, 5 parts～10 parts of EDTAP dipotassium ethylene diamine tetraacetate or two edetate disodium.The cell-preservation liquid patent of some subsequent application also, but all exist the cell holding time short, sample impurity is processed undesirable, and the interference sectioning cells factors such as haemocyte, mucus mucus are processed the defective such as comply with one's wishes not to the utmost.
Summary of the invention
Goal of the invention: the objective of the invention is in order to solve above-mentioned the deficiencies in the prior art, and provide that a kind of cell storage rate is high, holding time length, haemocyte, mucus treatment effect is good, the exfoliated cell preservative fluid that cost performance is higher.
Technical scheme: the present invention solves the problems of the technologies described above the technical scheme that adopts and is: a kind of exfoliated cell preservative fluid, the osmotic pressure of it is characterized in that comprising the pH buffer, keeping ion strength are kept agent, antisepsis antistaling agent, are used for keeping fixative, anticoagulant, mucus softening agent, antimicrobial reagent, humectant, the cleaning agent of cellular morphology and destroying erythrocytic composition.
As preferably, described pH buffer pH value is 6.8～7.4.
As preferably, described pH buffer is one or more in PBS, Tris-HCl or PIPES;
It is one or more in the solution such as sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate that described osmotic pressure is kept agent;
Described fixative is one or more in absolute ethyl alcohol, methyl alcohol, paraformaldehyde, acetone, coumarin;
Described anticoagulant is that ethylenediamine tetra-acetic acid two is received, one or more in EDTAP dipotassium ethylene diamine tetraacetate, heparin;
Described mucus softening agent is to draw together one or more in methyl cysteine, dithiothreitol (DTT) or dihydroxy dithiothreitol (DTT);
The penicillin of antimicrobial reagent 0.01%～0.5% or streptomycin;
Described humectant is at least a in phenol, glycerine;
Cleaning agent 0.1%～0.5%; Comprise one or more in nonionic, cationic, anionic or two property ionic surfactant.
described nonionic surface active agent comprises PVP K30, the APEO sim alkylphenol, one or more in Tween-20 or Tween-80, described cationic surface active agent comprises hexadecyldimethyl benzyl ammonium ammonium chloride, OTAC, cation guar gum, the cation panthenol, one or more in cation silicone oil, described anionic surfactant comprises sodium n-alkylbenzenesulfonate, one or more in lauryl sodium sulfate, described pair of property ionic surfactant comprises dodecyldimethylammonium hydroxide inner salt, in the carboxylic acid type imidazoline one or more.
The erythrocytic composition of described destruction comprises at least a in tertiary sodium phosphate, glacial acetic acid, bromo 16 alkyl trimethylamines.
As preferably, comprise the component of following percentage by weight:
And, the component of following percent by volume:
Wherein, described anticoagulant is that ethylenediamine tetra-acetic acid two is received, a kind of in EDTAP dipotassium ethylene diamine tetraacetate, heparin, the penicillin of antimicrobial reagent 0.01%～0.5%, streptomycin etc. a kind of, described pH buffer is a kind of in PBS, Tris-HCl or PIPES.
Beneficial effect: compared with prior art, the invention has the advantages that: preserve each component rational proportion in liquid, preserve longlyer under normal temperature, the longlyest can reach 2 years; Can fully dissolve mucus and partial destruction red blood cell; It is very even that production effect has cell distribution, and cellular morphology is intact, and endochylema and cell nucleus boundary are clear, well arranged, and the bright property of endochylema and cell nucleus is very good, can be used for simultaneously HPV-DNA, Chlamydia and SABC test; The special-purpose liquid base that can be provided for other position cytolgical examinations is preserved liquid, and the complete import substitutes of energy, has fully kept the cell component that diagnostic significance is arranged; Deployment cost is lower, is easy to promote.
Embodiment
In order to deepen the understanding of the present invention, the invention will be further described below in conjunction with embodiment, and this embodiment only is used for explaining the present invention, does not consist of the restriction to protection domain of the present invention.
Embodiment 1: in this example, cell used is cervical exfoliated cell, and flaking method is centrifugal settling method, and the exfoliated cell preservative fluid proportioning in the present embodiment is as follows:
Component 0.7% sodium chloride of percentage by weight, 0.25% tertiary sodium phosphate, 7.5% ethylenediamine tetra-acetic acid two is received, one of in EDTAP dipotassium ethylene diamine tetraacetate, heparin, 1% penicillin, 3% paraformaldehyde, 3% coumarin, 0.1% methyl cysteine, 0.3% glacial acetic acid, 2.5% carbolic acid, 0.2% Tween-20.
The component of percent by volume: 60%pH buffer (pH7), 16% absolute ethyl alcohol, 12.5% methyl alcohol, 3% acetone, wherein, the pH buffer is a kind of in PBS, Tris-HCl, PIPES.
Embodiment 2: this example cell used is cervical exfoliated cell, and flaking method is the membrane filtration method, and the exfoliated cell preservative fluid proportioning in the present embodiment is as follows:
The component of percentage by weight: 0.9% sodium chloride, 0.1% tertiary sodium phosphate, 10% ethylenediamine tetra-acetic acid two is received, one of in EDTAP dipotassium ethylene diamine tetraacetate, heparin, 1% streptomycin, 4% paraformaldehyde, 4% coumarin, 0.5% dithiothreitol (DTT), 0.5% bromo 16 alkyl trimethylamines, 1% glycerine, 0.5% OTAC.
The component of percent by volume: 50%pH buffer (pH6.8), 20% absolute ethyl alcohol, 15% methyl alcohol, 4% acetone, wherein, the pH buffer is a kind of in PBS, Tris-HCl, PIPES.
Embodiment 3: this example cell used is cervical exfoliated cell, and flaking method is natural sedimentation, and the exfoliated cell preservative fluid proportioning in the present embodiment is as follows:
The component of percentage by weight: 0.5% sodium chloride, O.5% tertiary sodium phosphate, 5% ethylenediamine tetra-acetic acid two is received, one of in EDTAP dipotassium ethylene diamine tetraacetate, heparin, 1% penicillin, 1% paraformaldehyde, 4% coumarin, O.3% dihydroxy dithiothreitol (DTT), 0.1% bromo 16 alkyl trimethylamines, 5% carbolic acid, 0.5% DDAO.
The component of percent by volume: 70%pH buffer (pH7.4), 12% absolute ethyl alcohol, 10% methyl alcohol, 2% acetone, wherein, the pH buffer is a kind of in PBS, Tris-HCl, PIPES.
Result of the test shows, the exfoliated cell preservative fluid cell storage rate of embodiment 1, embodiment 2 and embodiment 3 is high, cell distribution is very even, cellular morphology is intact, endochylema and cell nucleus boundary are clear, well arranged, the bright property of endochylema and cell nucleus is very good, be at same sample and all can well embody the Quality Control point when HPV-DNA detects, positive coincidence rate all has uniformity.
Holding time is long, all can preserve 2 years under normal temperature.
Claims (5)
1. exfoliated cell preservative fluid, it is characterized in that, comprise following composition: pH buffer, the osmotic pressure of keeping ion strength are kept agent, antisepsis antistaling agent, are used for keeping fixative, anticoagulant, mucus softening agent, antimicrobial reagent, humectant, the cleaning agent of cellular morphology and destroying erythrocytic composition.
2. a kind of exfoliated cell preservative fluid according to claim 1, it is characterized in that: described pH buffer pH value is 6.8～7.4.
3. a kind of exfoliated cell preservative fluid according to claim 1 is characterized in that:
Described pH buffer is one or more in PBS, Tris-HCl or PIPES;
It is one or more in the solution such as sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate that described osmotic pressure is kept agent;
Described fixative is one or more in absolute ethyl alcohol, methyl alcohol, paraformaldehyde, acetone, coumarin;
Described anticoagulant is that ethylenediamine tetra-acetic acid two is received, one or more in EDTAP dipotassium ethylene diamine tetraacetate, heparin;
Described mucus softening agent is to draw together one or more in methyl cysteine, dithiothreitol (DTT) or dihydroxy dithiothreitol (DTT);
The penicillin of described antimicrobial reagent 0.01%～0.5% or streptomycin;
Described humectant is at least a in phenol, glycerine;
Described cleaning agent is one or more in nonionic surface active agent, cationic surface active agent, anionic surfactant or two property ionic surfactant.
The erythrocytic composition of described destruction comprises at least a in tertiary sodium phosphate, glacial acetic acid, bromo 16 alkyl trimethylamines.
4. a kind of exfoliated cell preservative fluid according to claim 3, it is characterized in that: described nonionic surface active agent comprises PVP K30, the APEO sim alkylphenol, one or more in Tween-20 or Tween-80, described cationic surface active agent comprises hexadecyldimethyl benzyl ammonium ammonium chloride, OTAC, cation guar gum, the cation panthenol, one or more in cation silicone oil, described anionic surfactant comprises sodium n-alkylbenzenesulfonate, one or more in lauryl sodium sulfate, described pair of property ionic surfactant comprises dodecyldimethylammonium hydroxide inner salt, in the carboxylic acid type imidazoline one or more.
5. a kind of exfoliated cell preservative fluid according to claim 4 is characterized in that: the component that comprises following percentage by weight:
And, the component of following percent by volume:
Wherein, described anticoagulant is that ethylenediamine tetra-acetic acid two is received, a kind of in EDTAP dipotassium ethylene diamine tetraacetate, heparin, the penicillin of antimicrobial reagent 0.01%～0.5%, streptomycin etc. a kind of, described pH buffer is a kind of in PBS, Tris-HCl or PIPES.
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| CN103704200A (en) * | 2013-10-25 | 2014-04-09 | 无锡灵锡医疗器械科技有限公司 | Method for antibacterial stabilization of cell tissue sample compositions and structure |
| CN104585162A (en) * | 2014-12-30 | 2015-05-06 | 李东 | Novel environment-friendly nontoxic liquid-based cell preservation liquid |
| CN104642300A (en) * | 2015-03-13 | 2015-05-27 | 上海御康医院有限公司 | Exfoliative cell sap base preserving fluid, method thereof for flaking and kit |
| CN104745670A (en) * | 2015-04-16 | 2015-07-01 | 黄小军 | Preparation method of cell chip and application of cell chip in tumour screening field |
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| CN105241726A (en) * | 2015-09-14 | 2016-01-13 | 黄小军 | Immunohistochemical staining method and applications of immunohistochemical staining method in cervical tumorous lesion screening |
| CN105994252A (en) * | 2016-06-22 | 2016-10-12 | 杭州海世嘉生物科技有限公司 | Liquid-based thin cell preservation liquid and preparation method thereof |
| CN106234354A (en) * | 2016-09-30 | 2016-12-21 | 上海市奉贤区中心医院 | A kind of cervix cell preservation solution |
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| CN108402033A (en) * | 2018-04-10 | 2018-08-17 | 南京福怡科技发展股份有限公司 | One kind having preprocessing function cervical exfoliated cell preservative fluid and application |
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