CN101363011A - Cervical exfoliated cell preservative fluid - Google Patents
Cervical exfoliated cell preservative fluid Download PDFInfo
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- CN101363011A CN101363011A CNA2008102000087A CN200810200008A CN101363011A CN 101363011 A CN101363011 A CN 101363011A CN A2008102000087 A CNA2008102000087 A CN A2008102000087A CN 200810200008 A CN200810200008 A CN 200810200008A CN 101363011 A CN101363011 A CN 101363011A
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Abstract
The invention relates to a cervical exfoliated cell preservative liquid, the preservative liquid comprises the following components with the contents (by weight): 20 percent to 50 percent of alcohols; 15 percent to 50 percent of anti-aggregation reagent; 5 percent to 10 percent of buffer solution; 1 percent to 20 percent of ion strength maintaining reagent; 0.01 percent to 0.5 percent of anti-microbial reagent; 0.1 to 5 percent of mucus dissolving reagent; and 0 to 0.5 percent of cleaning agent. Compared with the prior art, the preservative liquid can not only lead cells to maintain the shape in an in vitro liquid suspension environment, minimize the protein precipitation, dissolve larger protein substances, such as blood and mucus, and reduce the cell aggregation, but can also selectively eliminate or reduce red cells, effectively kill microbes, prevent the activity of reverse transcriptase and retain the integrity of nucleic acids and proteins for facilitating the later analysis; in addition, the preservative liquid can greatly reduce the costs of consumptive materials for the TCT detection, improve the sensitivity and the specificity of the cervical cancer screening and accelerate the promotion and the popularization of the TCT technology in a medical system.
Description
Technical field
The present invention relates to cell-preservation liquid, relate in particular to a kind of cervical exfoliated cell preservative fluid.
Background technology
As the screening method of cervical lesions, what relate in traditional Pap smear process draws materials, is coated with all multifactor positive rates that all can seriously reduce cell such as tablet quality, cellular segregation.Cell engineering expert has released a kind of film-making new technology---and the liquid based thin-layer cell learns a skill (TCT), and it mainly loses non-diagnosis impurity by technical finesse, makes and observes thin-layer cell sheet clearly, makes the easier observation of person of readding the sheet, and diagnostic accuracy obviously improves.
The liquid based thin-layer cell learn a skill (TCT) changed the working method of Pap smear in the past, sample washes in the cell-preservation liquid after taking out immediately, and the cell of having avoided being retained in the conventional Pap smear process on the sampler is abandoned and cell transition exsiccant phenomenon with sampler.The cell of preserving in the liquid is handled through sequencing, uniform thin smear is made in random sampling, undesired cell in thin slice is observed easily, and wet fixed nucleus clear in structure, be easy to differentiate, can detect more rudimentary pathology and some more serious pathologies, be the new technology of relatively carrying out in the world at present.Adopt the liquid based thin-layer cell to learn a skill paracytic diagnosis has been improved 13%, the recall rate of the above pathology of low tesselated epithelium has been improved 65%, have the incomparable advantage of traditional smear.
The most key reagent is cell-preservation liquid in the TCT technology.At present, 40-50% alcohols commonly used is as preserving liquid on the market, in transportation and microcosmic slide storage, be used for sustenticular cell, can fix mobile high protein greater than 50%, but protein just forms settling immediately, can not change into slide, this brings difficulty to cytolgical examination, can cause the cell surface distortion simultaneously.If preservation can not be fixed for a long time, easily cause cell degradation less than 20% cell.
The TCT technology has become one of best recommend method of examination cervical cancer, for the early diagnosis and therapy of cervical cancer provides very clear and definite diagnosis basis, especially apply widely in today of modern civilization technology high-speed development and just seem particularly urgent and important, it is extremely urgent therefore to develop suitable cervical exfoliated cell preservative fluid.
Summary of the invention
Purpose of the present invention is exactly the cervical exfoliated cell preservative fluid that provides in order to overcome the defective that above-mentioned prior art exists that a kind of cell fixation is good, cost is lower, improves examination sensitivity and specific degree.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of cervical exfoliated cell preservative fluid is characterized in that, this preservation liquid comprises following component and content (weight):
Alcohols 20%-50%;
The anti-reagent 15%-50% that assembles;
Damping fluid 5%-10%;
Ionic strength is kept reagent 1%-20%;
Antimicrobial reagent 0.01%-0.5%;
Mucolysis reagent 0.1-5%;
Sanitising agent 0-0.5%.
Described alcohols comprises methyl alcohol, ethanol, Virahol; Described anti-gathering reagent comprises ethylenediamine tetraacetic acid (EDTA), edetate, liver kinases, streptokinase.
The first-selected disodium ethylene diamine tetraacetate of described edetate.
Described damping fluid comprises that acetic acid-acetate buffer, N-(2-kharophen)-2-aminoethyl sulfonic acid (ACES), N-(2-ethanamide) iminodiethanoic acid (ADA), two (2-hydroxyethyl)-Tutofusin triss (BIS-TRIS), 2-(N-morphine quinoline) ethyl sulfonic acid (MES), piperazine attain-N, and N '-two (2-ethanesulfonic acid) (PIPES); Described ionic strength is kept reagent and is comprised Repone K.
Described acetate is selected from one or more in sodium-acetate, magnesium acetate, the calcium acetate.
Described antimicrobial reagent comprises microbiotic; Described mucolysis reagent comprises that disulfide linkage opens reagent.
Described microbiotic comprises penicillin, Streptomycin sulphate, tsiklomitsin or penbritin.
Described disulfide linkage is opened reagent and is comprised methyl halfcystine, N-ethanoyl-L-halfcystine, dithiothreitol (DTT) or dihydroxyl dithiothreitol (DTT).
Described sanitising agent comprises non-ionic type, cationic, anionic or two property ionogenic surfactant.
Described nonionic surface active agent comprises polyvinylpyrrolidone K30, the Soxylat A 25-7 sim alkylphenol, tween 20 or tween-80, described cationic surfactant comprises hexadecyldimethyl benzyl ammonium ammonium chloride, octadecyl trimethyl ammonium chloride, cation guar gum, the positively charged ion panthenol, positively charged ion silicone oil or dimethyl dodecyl amine oxide, described aniorfic surfactant comprises linear alkylbenzene sulphonic acid, α-sodium olefin sulfonate, polyoxyethylenated alcohol sodium sulfate or sodium lauryl sulphate, described pair of property ionogenic surfactant comprises Varion CDG-K or carboxylic acid type tetrahydroglyoxaline.
First composition alcohols among the present invention can be kept cell DNA and protein integrity, and first-selected methyl alcohol also can be selected ethanol, Virahol for use, and ratio is 20-50%;
Second composition is the anti-reagent of assembling, and selects the anti-gathering reagent that can be dissolved in methyl alcohol or other alcohols for use, comprises that huge legendary turtle closes reagent-EDTA, and first-selected disodium bicarbonate synthetic EDTA also can select liver kinases, streptokinase etc. for use, and concentration approximately is: 15%-50%;
The third composition is a damping fluid, be used for regulating the pH value, adapting to the pH that cell release self-dissolving by product is caused in the sample changes, to keep cell morphological characteristic, first-selected acetate buffer, as sodium-acetate, magnesium acetate, calcium acetate or associating, also can select for use other as N-(2-kharophen)-2-aminoethyl sulfonic acid (ACES), N-(2-ethanamide) iminodiethanoic acid (ADA), two (2-hydroxyethyl)-Tutofusin triss (BIS-TRIS), 2-(N-morphine quinoline) ethyl sulfonic acid (MES), piperazine is attained-N, N '-two (2-ethanesulfonic acid) (PIPES), concentration is approximately 5%-10%;
The 4th kind of composition is in order to keep ionic strength, to prevent the cell modification, not only to be dissolved in to preserve and to produce anti-freezing reagent (EDTA) precipitation in the liquid, select Repone K for use, concentration is approximately 1%-20%, the material of keeping ionic strength formerly additionally adds in the solution, or is compatible with in the damping fluid in addition;
The 5th kind of composition is antimicrobial reagent, in order to the microorganism in the preservation liquid that suppresses sample to be tested: Candida albicans, aspergillus tubigensis, intestinal bacteria, pseudomonas and staphylococcus, the factor that consideration was degraded with collection, transportation, analysis time is chosen microbiotic, comprise penicillin, Streptomycin sulphate, tsiklomitsin or penbritin etc., antibiotic content approximately is 0.01%-0.5%;
The 6th kind of composition is mucolysis reagent, comprise methyl halfcystine, N-ethanoyl-L-halfcystine, dithiothreitol (DTT) (DTT, a kind of protein denaturant), dihydroxyl dithiothreitol (DTT) or other can break the reagent of disulfide linkage, concentration is 0.1%-5%;
In addition, the present invention preserves and also contains sanitising agent in the liquid, comprises non-ionic type, cationic, anionic or two property ionogenic surfactant.
Compared with prior art, preservation liquid of the present invention not only can make cell can keep form in external liquid suspension environment, minimize albumen precipitation, dissolve bigger proteic substance such as blood, mucus, reduce cell aggregation, but also can selectivity eliminate or minimizing red corpuscle, effectively killing microorganisms, stop reverse transcriptase activity, it is complete so that with post analysis to keep nucleic acid and albumen; In addition, the present invention reduces the consumables cost that TCT detects greatly, has improved the sensitivity and the specific degree of cervical cancer examination, has quickened TCT technology popularizing in medical system.
Description of drawings
Fig. 1 is the synoptic diagram of the Pathologic specimen of employing the present invention making;
Fig. 2 is for adopting first kind of existing synoptic diagram of preserving the Pathologic specimen of liquid making;
Fig. 3 is for adopting second kind of existing synoptic diagram of preserving the Pathologic specimen of liquid making.
Embodiment
The invention will be further described for the contrast drawings and the specific embodiments below.
Embodiment 1
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols methyl alcohol | 20 |
| The anti-reagent disodium ethylene diamine tetraacetate of assembling | 49.89 |
| Damping fluid acetic acid-sodium-acetate buffer | 10 |
| Ionic strength is kept reagent Repone K | 20 |
| Antimicrobial reagent penicillin | 0.01 |
| Mucolysis reagent methyl halfcystine | 0.1 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 2
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols ethanol | 30 |
| The anti-reagent liver kinases of assembling | 38.9 |
| Damping fluid N-(2-kharophen)-2-aminoethyl sulfonic acid (ACES) | 10 |
| Ionic strength is kept reagent Repone K | 20 |
| The antimicrobial reagent Streptomycin sulphate | 0.1 |
| Mucolysis reagent N-ethanoyl-L-halfcystine | 1 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 3
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| The alcohols Virahol | 35 |
| The anti-reagent streptokinase of assembling | 39.3 |
| Damping fluid N-(2-ethanamide) iminodiethanoic acid (ADA) | 8 |
| Ionic strength is kept reagent Repone K | 15 |
| The antimicrobial reagent tsiklomitsin | 0.2 |
| Mucolysis reagent dithiothreitol (DTT) | 2 |
| Sanitising agent α-sodium olefin sulfonate | 0.5 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 4
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols methyl alcohol | 40 |
| The anti-reagent streptokinase of assembling | 28.4 |
| Two (2-the hydroxyethyl)-Tutofusin triss (BIS-TRIS) of damping fluid | 8 |
| Ionic strength is kept reagent Repone K | 20 |
| The antimicrobial reagent penbritin | 0.3 |
| Mucolysis reagent dihydroxyl dithiothreitol (DTT) | 3 |
| The sanitising agent Varion CDG-K | 0.3 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 5
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols methyl alcohol | 45 |
| The anti-reagent streptokinase of assembling | 27.4 |
| Two 2-(the N-morphine quinoline) ethyl sulfonic acids (MES) of damping fluid | 8 |
| Ionic strength is kept reagent Repone K | 15 |
| The antimicrobial reagent penbritin | 0.4 |
| Mucolysis reagent methyl halfcystine | 4 |
| The sanitising agent tween 20 | 0.2 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 6
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols methyl alcohol | 50 |
| The anti-reagent streptokinase of assembling | 19.5 |
| The two piperazines of damping fluid are attained-N, and N '-two (2-ethanesulfonic acid) (PIPES) | 10 |
| Ionic strength is kept reagent Repone K | 15 |
| The antimicrobial reagent penbritin | 0.5 |
| Mucolysis reagent dithiothreitol (DTT) | 5 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 7
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols methyl alcohol | 50 |
| The anti-reagent disodium ethylene diamine tetraacetate of assembling | 40.6 |
| Damping fluid acetic acid-sodium-acetate buffer | 5 |
| Ionic strength is kept reagent Repone K | 1 |
| Antimicrobial reagent penicillin | 0.3 |
| Mucolysis reagent methyl halfcystine | 3 |
| Sanitising agent polyvinylpyrrolidone K30 | 0.1 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
Embodiment 8
A kind of cervical exfoliated cell preservative fluid, this preservation liquid comprise following component and content (weight):
| Component | Content (Kg) |
| Alcohols ethanol | 50 |
| The anti-reagent liver kinases of assembling | 15 |
| Damping fluid N-(2-kharophen)-2-aminoethyl sulfonic acid (ACES) | 10 |
| Ionic strength is kept reagent Repone K | 19 |
| The antimicrobial reagent Streptomycin sulphate | 0.5 |
| Mucolysis reagent N-ethanoyl-L-halfcystine | 5 |
| Sanitising agent hexadecyldimethyl benzyl ammonium ammonium chloride | 0.5 |
Said components is obtained a kind of cervical exfoliated cell preservative fluid according to the content requirement uniform mixing.
By the design double blind experiment the present invention is carried out quality evaluation, specific as follows:
The section random collecting uses this product and the present Pathologic specimen made of other two kinds of cell-preservation liquids (hereinafter replacing with B liquid and C liquid respectively) of generally using of tertiary hospitals in the recent period at one time.All slice, thin pieces are made by pathology chamber, Ai Dikang Clinical Laboratory center, collect 300 pathology sheets altogether, comprise 100 of this product A liquid, 105 of B liquid, 95 of C liquid.
The patent applicant invites the most experienced TCT diagnostician in Zhejiang Province TCT preservation liquid to three groups of different labels under the prerequisite of not knowing producer's brand to assess, and to reach just objective purpose, assessment result is as follows:
Evaluation index: total background, general cell amount, cellular form and preservation quality are to the influence of diagnosis.
Assessment result:
A organizes (the present invention): as shown in Figure 1, the general cell amount still can, cell is preserved good, lymphocyte exists preservation not good, it is harder to observe pathologies such as trichomonad, bleb, is applied to the clinical positive rate and the accuracy rate that can influence diagnosis to a certain extent, but belongs to a kind of preferably in three's the comparison;
(annotate: B liquid): as shown in Figure 2, cell concentration is preceding relatively a kind of less, preserves good for the B group, a kind of before being better than, but background is more random, and cell regression situation is also more common, can influence the positive rate and the accuracy rate of diagnosis to a certain extent, overall and the former (being the present invention) is more or less the same;
C group (annotate: C liquid): as shown in Figure 3, the three relatively in this group preservation effect the poorest, cellular swelling is obvious, the slice, thin piece amount of influence diagnosis is a lot, does not advise being applied to the laboratory.
The present invention can be packaged as 20 milliliters/bottle, is used for the preservation of cervical exfoliated cell, adopts the liquid based thin-layer cell to learn a skill and makes the cervical exfoliated cell slice, thin piece, carries out cytodiagnosis for the pathology doctor.
The liquid based thin-layer cell is learned detection system (Thinprep Cytologic Test, TCT) main method is cervical exfoliated cell to be put into the bottle of preserving liquid is housed, by the film-making of centrifugation technology, make diagnosis report by the TBS method again, this method is than traditional smear method accuracy rate height.
Cervical exfoliated cell preservative fluid of the present invention can play fixed cell, prevents cell aggregation, disperses mucus, removes red corpuscle, keeps pH value and ionic strength, prevents cytomorphosis, effects such as normal temperature preservation.
Use sampling store method of the present invention to be:
The 1st step: besom sample Uterine neck bush is inserted endocervical canal and light the pressure, and bristle coincide the external os of vervix and tilts;
The 2nd step: keep pressure, thumb and forefinger are held handle, and hairbrush 5-8 time turns clockwise;
The 3rd step: uterine cervix brush is inserted in the liquid base bottle, stir about 10 times, and powerful rotation bristle abandons brush at last;
The 4th step: tighten the lid of preserving the liquid bottle;
The 5th step: on body, indicate patient name, medical record information etc. and inspect by ready samples with request slip.
Claims (10)
1. a cervical exfoliated cell preservative fluid is characterized in that, this preservation liquid comprises following component and content (weight):
Alcohols 20%-50%;
The anti-reagent 15%-50% that assembles;
Damping fluid 5%-10%;
Ionic strength is kept reagent 1%-20%;
Antimicrobial reagent 0.01%-0.5%;
Mucolysis reagent 0.1-5%;
Sanitising agent 0-0.5%.
2. cervical exfoliated cell preservative fluid according to claim 1 is characterized in that described alcohols comprises methyl alcohol, ethanol, Virahol; Described anti-gathering reagent comprises ethylenediamine tetraacetic acid (EDTA), edetate, liver kinases, streptokinase.
3. cervical exfoliated cell preservative fluid according to claim 2 is characterized in that, the first-selected disodium ethylene diamine tetraacetate of described edetate.
4. cervical exfoliated cell preservative fluid according to claim 1, it is characterized in that, described damping fluid comprises that acetic acid-acetate buffer, N-(2-kharophen)-2-aminoethyl sulfonic acid (ACES), N-(2-ethanamide) iminodiethanoic acid (ADA), two (2-hydroxyethyl)-Tutofusin triss (BIS-TRIS), 2-(N-morphine quinoline) ethyl sulfonic acid (MES), piperazine attain-N, and N '-two (2-ethanesulfonic acid) (PIPES); Described ionic strength is kept reagent and is comprised Repone K.
5. cervical exfoliated cell preservative fluid according to claim 4 is characterized in that described acetate is selected from one or more in sodium-acetate, magnesium acetate, the calcium acetate.
6. cervical exfoliated cell preservative fluid according to claim 1 is characterized in that described antimicrobial reagent comprises microbiotic; Described mucolysis reagent comprises that disulfide linkage opens reagent.
7. cervical exfoliated cell preservative fluid according to claim 6 is characterized in that described microbiotic comprises penicillin, Streptomycin sulphate, tsiklomitsin or penbritin.
8. cervical exfoliated cell preservative fluid according to claim 6 is characterized in that, described disulfide linkage is opened reagent and comprised methyl halfcystine, N-ethanoyl-L-halfcystine, dithiothreitol (DTT) or dihydroxyl dithiothreitol (DTT).
9. cervical exfoliated cell preservative fluid according to claim 1 is characterized in that, described sanitising agent comprises non-ionic type, cationic, anionic or two property ionogenic surfactant.
10. cervical exfoliated cell preservative fluid according to claim 9, it is characterized in that, described nonionic surface active agent comprises polyvinylpyrrolidone K30, the Soxylat A 25-7 sim alkylphenol, tween 20 or tween-80, described cationic surfactant comprises hexadecyldimethyl benzyl ammonium ammonium chloride, octadecyl trimethyl ammonium chloride, cation guar gum, the positively charged ion panthenol, positively charged ion silicone oil or dimethyl dodecyl amine oxide, described aniorfic surfactant comprises linear alkylbenzene sulphonic acid, α-sodium olefin sulfonate, polyoxyethylenated alcohol sodium sulfate or sodium lauryl sulphate, described pair of property ionogenic surfactant comprises Varion CDG-K or carboxylic acid type tetrahydroglyoxaline.
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| CNA2008102000087A CN101363011A (en) | 2008-09-17 | 2008-09-17 | Cervical exfoliated cell preservative fluid |
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|---|---|---|---|
| CNA2008102000087A CN101363011A (en) | 2008-09-17 | 2008-09-17 | Cervical exfoliated cell preservative fluid |
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| CN101363011A true CN101363011A (en) | 2009-02-11 |
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