CN1967193A - Cell processing kit and using method therefor - Google Patents
Cell processing kit and using method therefor Download PDFInfo
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- CN1967193A CN1967193A CN 200510110535 CN200510110535A CN1967193A CN 1967193 A CN1967193 A CN 1967193A CN 200510110535 CN200510110535 CN 200510110535 CN 200510110535 A CN200510110535 A CN 200510110535A CN 1967193 A CN1967193 A CN 1967193A
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Abstract
The invention provides a cell processing reagent box for cell detection and its usage method, the reagent box including the specimens preserved fluid, the separating and cleaning fluid, the packet diluted base liquid, and also the cell reserved liquid. Using the reagent box to process the cells, the operation is simple and fast, no need special equipments, low-cost, and good cells processing effect, which will help enhance the accuracy rate of detection, and can be widely applied to the clinic and laboratory.
Description
Technical field
The present invention relates to a kind of cell processing kit and using method thereof, relate in particular to a kind of treatment kits and using method thereof that is used for clinical detection with cell specimen.
Technical background
Cyto-diagnosis also claims cell pathology or clinical cytology, be that cell is made cell smear, through cytochemistry or immunocytochemistry colour developing, based on cytology, histopathology is for instructing, observe various normal and pathologic cells in cell smear, its lesion nature of analysis-by-synthesis reaches and diagnoses the illness or the purpose of pathology.Cytodiagnosis can be used for the inspection of various cast-off cells in female genital tract, esophagus, respiratory tract, ascites, the urine etc., also can be used for the cell that fine needle aspiration such as mammary gland, liver obtain, is very extensive and simple and easy to do, the cost-effective pathological diagnosis method of a kind of clinical practice.
The uterus is the distinctive vitals of women, and it is divided into palace body and uterine neck two parts, and the palace body is positioned at pelvic cavity, and uterine neck is positioned at vagina, and uterine neck and female reproduction activity and sexual life are closely related.Because its function and present position, cervix lesion is one of modal illness of women.According to The World Health Organization (WHO), annual de novo cervical carcinoma patient 450,000 people in the whole world.According to statistics, if doing once conventional Pasteur in per 3 years, all women check that the cervical carcinoma incidence of disease will descend 83%; If use the liquid based cytology inspection, the cervical carcinoma incidence of disease will be checked decline 92% than not doing.Reason be cervical carcinoma exist one by the reversible precancerous lesion phase to carcinoma in situs of cervix and then develop into uterine neck and invade the continuous evolution of moistening cancer, these forerunner's pathologies can exist for many years, and cervix has favourable anatomical location, be easy to expose, be convenient to observation, palpation and draw materials, cervical carcinoma if can be made a definite diagnosis treatment in the cercinoma prophase pathologic change stage, and cervical carcinoma can not only be prevented, and can effect a radical cure fully.
The inspection method of cervical lesions is concluded: 1. macroscopical indagation method: the visual inspection of gynaecology, vaginoscopy, uterine neck photograph, fluorescence microscopy, palpation etc.; 2. microstructure cytolgical examination method: the microexamination of cervical cytology smear, biological tissue pathological examination (biopsy) 3. virology detect.
Macroscopic view indagation method is not easy to judge its essence for early lesion; Though the essence of disease can be accurately concluded in biopsy, clamp and get the uterine neck living tissue, and uterine neck is caused damage, and scope of selecting material is very limited; Though epidemiology and biological information proof human papilloma virus (HPV) infect may be cervical carcinoma and precancerous lesion thereof main diseases because of, but not unique reason, not all cervical carcinoma causes also do not have evidence to show that it is cervical carcinoma that HPV infects inevitable development by virus.
The cervical cytology plate coating checking is to not damage of human body, patient's no pain, and cost is low, scope of selecting material comprises whole uterine neck, can observe directly the lesion degree and the essence of cellular level, its vital role is generally acknowledged by common people that already its maximum value part also is energy early diagnosis preclinical phase cervical carcinoma, thereby be the one preferred technique method of gynaecology's outpatient service to cervical carcinoma examination and eliminating, examination regularly also is the unique method of present whole world prevention cervical carcinoma.
The cervical cytology smear technique is that the mucus that contains cast-off cells of will stick on the cervical cell collector directly is applied on the microslide at first, dyed back microscopic examination.This method is by Aretaeus of Cappadocia Papanicolaou invention, thereby is called " Pap smear method (Pap Smear) ", begin to introduce the forties in last century as a kind of means of cervical carcinoma examination clinical, afterwards by many countries as conventional examination project.Because of its method is easy, patient's no pain, and cost is lower, being fit to very much crowd's generaI investigation on a large scale, almost having been continued to use nearly half a century so far unalterablely, is the 3rd most widely used detection system in the world, be only second to diabetes and malaria, is still using in some place.
But the eighties intermediary and later stages, the cervical smear that occurs in the U.S. is diagnosed the report 3 of false negative case, has caused people's shock.When the specificity of cervical cytology diagnosis and susceptibility are analyzed, find that all there is appreciable misdiagnosis rate 4 the different experiments chamber.In self-examination, it should be noted that the following three aspect reasons of mainly containing of false negative case to the Pap smear technology.
1. the defective of Pap smear method:
1. misdiagnosis rate height: being early cervical carcinoma originally but can not finding, is false negative, makes cancer development to late period, causes death.The false negative of tradition Pap smear method can reach 5%-30%.Below be the false negative he result of investigation of delivering in recent years about traditional Pap smear:
| The author | False negative result number percent |
| Gay, etc. (1985) | 19.5 |
| Van der Graaf, etc. (1987) | 17.0 |
| Giles, etc. (1988) | 32.0 |
| Mitchell, etc. (nineteen ninety) | 13.2 |
| Joseph, etc. (1991) | 11.0 |
| Kristensen, etc. (1991) | 30.9 |
| Soost, etc. (1991) | 20.0 |
| Average false negative | 20.6 |
2. susceptibility is low: because the restriction of traditional Pap smear detection sensitivity, detecting the efficient of cervical carcinoma only is 51%.
3. false positive height and poor repeatability: the poor repeatability of detection causes result's instability, occurs false positive easily, not only can allow patient bear the cost that a unnecessary nearly step checks, but also patient is caused moral injury.
3. the Pap smear method causes the technical reason of defective:
Main cause is the mistake that the intrinsic sample disposal defective of this method itself causes.
(1) collect and to have only 10% to 30% cell can transfer on the microslide on the utensil, remaining cell all is abandoned, thereby sick cell may be missed, and can not reflect pathology comprehensively;
(2) smear can not be dried, and should fix with immobile liquid in 15 to 30 seconds, but be difficult in the real work accomplish, thereby be easy to generate the artefact of cytomorphosis;
(3) too much mucus, red blood cell, leucocyte etc. on the smear are given and are read sheet and cause difficulty, are easy to generate to read tiredly, are difficult for finding sick cell;
(4) cast-off cells accumulation, overlapping makes abnormal cell covered, and this all can have a strong impact on cell observation, is difficult to make accurate judgement.
At these problems, the cytologic slide new technology constantly produces and development, and traditional Pap smear method is stopped using gradually, the substitute is modern uterine neck liquid based cytology tabletting technology.
Cell pathology diagnosis comprises three continuous processes: film-making, dye, read sheet, film-making is whole cytopathologic basis, be can be correctly the condition precedent of diagnosis down.Liquid basal cell (the Liquid Based Cytology that learns a skill, LBC) mainly be meant tabletting technology in the cell pathology, be that the cast-off cells sample that will collect on the utensil of drawing materials is all transferred in the cell-preservation liquid, remove irrelevant cell, mucus and other interference impurity by various particular processing methods, make thin-layer cell smear clearly.Be the revolution of modern cytologic specimen method for making, its advantage is:
(1) almost keeps the whole samples on the sampler, be difficult for the omission sick cell, reduced false-negative generation.
(2) artefact who has avoided the cell transition drying to cause has reduced false-positive appearance.
(3) removed mucus too much on the smear, red blood cell, leucocyte etc. and the irrelevant disturbing factor of diagnosis, be convenient to machine recognition, the manual read has improved and has read sheet efficient, alleviates the person's that reads the sheet fatigue, makes to read sheet and more be difficult for makeing mistakes.
(4) cell in the thin smear do not pile up, overlapping, impurity is few, background clearance, cell image is clear, undesired cell is observed easily, is easy to differentiate, has improved the susceptibility that detects.
Since last century Mo, this method occurred, developed two generation technique products:
The first generation: filtering membrane isolation technics cell smear system
Its representative products is that Cytyc company 1996 is applied to clinical Xin Bai (ThinprepTM claims TCT again).
Main method is that cervical exfoliated cell is washed in the bottle of cell-preservation liquid, the scraping blade hairbrush stirred for ten seconds in bottle, again after filtering by filtering membrane on the machine of the said firm, impurity in the sample is separated, get epithelial cell after the filtration make diameter be the 20mm thin-layer cell on microslide, 95% alcohol fixation is through pap staining, mounting, the expert with the naked eye reads sheet at microscopically by cytology, makes diagnosis report by the TBS method.
The second generation: centrifugation technology cell smear system
Its product is the Suo Bai (Surpath that Tripath company entered clinical use in 1999
TM, claim LCT again).
Main method is that cervical exfoliated cell is washed in the bottle of cell-preservation liquid, machine proportion with the said firm is centrifugal, mucus in the sample, blood and inflammatory cell are separated, and it is that the 13mm thin-layer cell is on microslide that the remaining epithelial cell of collection is made diameter by machine.Each mechanical automation is finished, and can handle 48 parts of samples simultaneously, finishes cell dyeing simultaneously.
But these technology all are to rely on machine to finish at present, have machine operation complexity, expensive shortcoming, have improved medical treatment cost and patient burden undoubtedly.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of cell processing kit and using method thereof, to solve the defective that exists in the prior art.
One of purpose of the present invention is to provide a kind of cell processing kit, and this kit comprises following several reagent:
A. sample is preserved liquid and is formed: the isopropyl alcohol, 9.8% sodium chloride and the Tris damping fluid of 10-40% that comprise hexanol, the 30-50% of following components in weight percentage: 30-50%; Effect is to be used for fixing, to preserve sample;
B. separation cleaning fluid: contain the saccharoSan of 5-30% and 9.8% sodium chloride, be used for cleaning sample;
C. wrap diluted base fluid: be the poly-D-lysine aqueous solution of 0.005-0.02%, its effect is to be used for diluting and cohering cell on microslide.
This kit also comprises a kind of cell storing solution, and its composition is the hexanol of 50-80%, is used to remain the preservation inspection fully of sample.
Another object of the present invention has provided the using method of this kit, and this method comprises the steps:
A. the sample of collecting being dropped into immediately sample preserves in the liquid;
B. 4 milliliters of separation cleaning fluids will be added in the centrifuge tube;
C. sample was detained in preserving liquid after 30 minutes, sample is preserved the liquid bottle place whirlpool vortex mixer vibration 3 minutes, and abundant suspendible is poured the centrifuge tube of inclination tube wall into, makes sample preserve liquid on the liquid level of separation cleaning fluid;
D. centrifuge tube was placed the centrifugal 5-15 of hydro-extractor minute;
E. take out centrifuge tube, topple over supernatant at once;
F. in containing the centrifuge tube of cell precipitation, add 1-3 doubly to the diluted base fluid of bag of precipitation residue, place whirlpool vortex mixer vibration 10-20 second;
G. draw 50 microlitre cell suspensions with sample injector, the suction nozzle tip of pipettor is placed on the dry microslide of crossing with soaked in absolute ethyl alcohol gently, sample is painted 1～2 centimetre liquid film, drying gets final product.
Long preservation sample if desired will add 5-10ml physiological saline, vortex oscillator vibration 0.5-1 minute in the resultant centrifuge tube of toppling over behind the updip liquid in the step e, with remaining sample washing, centrifugal 5-10 minute, abandon supernatant, add cell storing solution 1-3ml, mixing moves into the EP pipe and seals up for safekeeping.
This kit is that a kind of cost is low, uses product easy to operate, that cell detection product, its use efficiently do not need specific installation to invest, and more hospitals can use this product, and more people can afford to use this product.
The overall advantage of this kit is summarized as follows:
| Technical characterstic | Tell on | Exert an influence |
| Up-to-date cell treatment technology | Clean cell background, perfect cellular morphology, the readable enhancing | Increase diagnosis person's confidence, improve diagnosis, alleviate psychological pressure and labour intensity |
| The reagent composition of nonhazardous | Harmless to human body | The working environment of safety and Health |
| Full sample is preserved | Cell is all preserved, and is convenient to deposit and transport | More sick cell occurs, and the film-making time is flexible |
| Extremely easy operating process | Save time fast, | Be easy to grasp, need not Special Training |
| The compatibility of height | Unite technology such as utilization immunocytochemistry, in situ hybridization, PCR | Need not repeatedly repeated acquisition sample |
| Need not specific installation | Only need generic centrifuge and common application of sample rifle | Easily have global state-of-the-art new technology |
Embodiment
Embodiment 1:
1. sample is preserved the liquid composition:
Comprise: hexanol: isopropyl alcohol 40%: sodium chloride 40%: Tris damping fluid 9.8%: 10%;
2. separation cleaning fluid: be 20% saccharoSan and 9.8% sodium chloride solution;
3. wrap diluted base fluid: principal ingredient is 0.01% poly-D-lysine aqueous solution;
4. cell storing solution: principal ingredient is 70% hexanol.
Embodiment 2:
The using method of cell processing kit comprises the steps:
A: the sample of collecting is dropped into sample immediately preserve in the liquid,, the brush input sample of sample brush is preserved liquid, screw bottle cap, transfer the film-making laboratory if use the separable specimen sampling brush of head.
B: will add 4 milliliters of separation cleaning fluids in the centrifuge tube.
C: sample was detained in preserving liquid after 30 minutes, sample is preserved the liquid bottle place whirlpool vortex mixer vibration 3 minutes, and abundant suspendible is poured the centrifuge tube of inclination tube wall gently into, makes sample preserve liquid on the liquid level of separation cleaning fluid.
D: centrifuge tube was placed hydro-extractor centrifugal 10 minutes, and a barrel formula centrifuge speed 900g wafts.
E: in hydro-extractor, take out centrifuge tube, topple over supernatant at once, and be inverted in suction and blot mouth of pipe remaining liq on the paper handkerchief.
F: in containing the centrifuge tube of cell precipitation, add the diluted base fluid of bag that doubles the precipitation residue approximately, place the whirlpool vortex mixer to vibrate for 15 seconds.
G: draw 50 microlitre cell suspensions with sample injector, the suction nozzle tip of pipettor is placed on the dry microslide of crossing with soaked in absolute ethyl alcohol gently, adopt the limit to draw the mode that limit, concentric garden pushes away the discharge opeing body sample is painted 1～2 centimetre liquid film, put in the room temperature dry.
H: will add 5 physiological saline in the centrifuge tube of toppling in the step e after the supernatant, vortex oscillator vibration one minute, with remaining sample washing, centrifugal 10 minutes of 900g abandons supernatant, adds cell storing solution 1ml, and mixing moves into EP pipe lid and tightly seals up for safekeeping.
Embodiment 3:
The using method of cell processing kit comprises the steps:
A: the brush that the sample of collecting is brushed drops into sample preservation liquid, screws bottle cap, transfers the film-making laboratory.
B: will add 4 milliliters of separation cleaning fluids in the centrifuge tube.
C: sample was detained in preserving liquid after 30 minutes, sample is preserved the liquid bottle place whirlpool vortex mixer vibration 3 minutes, and abundant suspendible is poured the centrifuge tube of inclination tube wall gently into, makes sample preserve liquid on the liquid level of separation cleaning fluid.
D: centrifuge tube was placed hydro-extractor centrifugal 10 minutes, bucket formula centrifuge speed 1100g.
E: in hydro-extractor, take out centrifuge tube, topple over supernatant at once, and be inverted in suction and blot mouth of pipe remaining liq on the paper handkerchief.
F: in containing the centrifuge tube of cell precipitation, add the diluted base fluid of bag that doubles the precipitation residue approximately, place the whirlpool vortex mixer to vibrate for 15 seconds.
G: draw 50 microlitre cell suspensions with sample injector, the suction nozzle tip of pipettor is placed on the dry microslide of crossing with soaked in absolute ethyl alcohol gently, adopt the limit to draw the mode that limit, concentric garden pushes away the discharge opeing body sample is painted 1～2 centimetre liquid film, place the baking oven that is no more than 50 ℃ dry.
H: will add 5 physiological saline in the centrifuge tube of toppling in the step e after the supernatant, vortex oscillator vibration one minute, with remaining sample washing, centrifugal 10 minutes of 1100g abandons supernatant, adds cell storing solution 1ml, and mixing moves into EP pipe lid and tightly seals up for safekeeping.
Claims (4)
1. cell processing kit is characterized in that this kit comprises following several reagent:
A. sample is preserved liquid: the isopropyl alcohol, 9.8% sodium chloride and the Tris damping fluid of 10-40% that comprise hexanol, the 30-50% of 30-50%;
B. separation cleaning fluid: comprise the saccharoSan of 10-60% and 9.8% sodium chloride;
C. wrap diluted base fluid: be the poly-D-lysine aqueous solution of 0.005-0.02%.
2. cell processing kit according to claim 1 is characterized in that also comprising the hexanol of 50-80%.
3. the using method of claim 1 or 2 described cell processing kits is characterized in that this using method comprises the steps:
A. the sample of collecting being dropped into immediately sample preserves in the liquid;
B. 4 milliliters of separation cleaning fluids will be added in the centrifuge tube;
C. sample was detained in preserving liquid after 30 minutes, sample is preserved the liquid bottle place whirlpool vortex mixer vibration 3 minutes, and abundant suspendible is poured the centrifuge tube of inclination tube wall into, makes sample preserve liquid on the liquid level of separation cleaning fluid;
D. centrifuge tube was placed the centrifugal 5-15 of hydro-extractor minute;
E. take out centrifuge tube, topple over supernatant at once;
F. in containing the centrifuge tube of cell precipitation, add 1-3 doubly to the diluted base fluid of bag of precipitation residue, place whirlpool vortex mixer vibration 10-20 second;
G. draw 50 microlitre cell suspensions with sample injector, the suction nozzle tip of pipettor is placed on the dry microslide of crossing with soaked in absolute ethyl alcohol gently, sample is painted 1～2 centimetre liquid film, drying gets final product.
4. the using method of cell processing kit according to claim 3, it is characterized in that also comprising the steps: and to add 5-10ml physiological saline in the resultant centrifuge tube of toppling over behind the updip liquid in the step e, vortex oscillator vibration 0.5-1 minute, with remaining sample washing, centrifugal 5-10 minute, abandon supernatant, add cell storing solution 1-3ml, mixing moves into the EP pipe and seals up for safekeeping.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510110535 CN1967193A (en) | 2005-11-18 | 2005-11-18 | Cell processing kit and using method therefor |
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| CN 200510110535 CN1967193A (en) | 2005-11-18 | 2005-11-18 | Cell processing kit and using method therefor |
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| CN1967193A true CN1967193A (en) | 2007-05-23 |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103471892A (en) * | 2013-09-22 | 2013-12-25 | 厦门大学附属第一医院 | Method for preparing pulled type cervical smear |
| CN104345142A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Technology for improving activity of coated perinuclear-factor antigen |
| 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 |
| 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 |
| CN107014658A (en) * | 2017-05-27 | 2017-08-04 | 四川省肿瘤医院 | Extract the method and kit of free cell |
| CN109030146A (en) * | 2018-07-25 | 2018-12-18 | 麦克奥迪（厦门）医疗诊断***有限公司 | A kind of sectioning cells reagent, kit and application method |
| CN111024471A (en) * | 2019-12-17 | 2020-04-17 | 浙江殷欣生物技术有限公司 | Liquid-based cell detection kit for cervical cancer screening and use method |
| CN112913834A (en) * | 2021-02-04 | 2021-06-08 | 杭州海世嘉生物科技有限公司 | Non-gynecological exfoliated cell preservation solution, kit containing preservation solution and application |
| WO2022078235A1 (en) * | 2020-10-16 | 2022-04-21 | 赵稳兴 | Automatic cell block preparation machine and method |
-
2005
- 2005-11-18 CN CN 200510110535 patent/CN1967193A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104345142A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Technology for improving activity of coated perinuclear-factor antigen |
| CN103471892A (en) * | 2013-09-22 | 2013-12-25 | 厦门大学附属第一医院 | Method for preparing pulled type cervical smear |
| 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 |
| 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 |
| CN107014658A (en) * | 2017-05-27 | 2017-08-04 | 四川省肿瘤医院 | Extract the method and kit of free cell |
| CN107014658B (en) * | 2017-05-27 | 2023-08-25 | 四川省肿瘤医院 | Method and kit for extracting free cells |
| CN109030146A (en) * | 2018-07-25 | 2018-12-18 | 麦克奥迪（厦门）医疗诊断***有限公司 | A kind of sectioning cells reagent, kit and application method |
| CN111024471A (en) * | 2019-12-17 | 2020-04-17 | 浙江殷欣生物技术有限公司 | Liquid-based cell detection kit for cervical cancer screening and use method |
| WO2022078235A1 (en) * | 2020-10-16 | 2022-04-21 | 赵稳兴 | Automatic cell block preparation machine and method |
| CN112913834A (en) * | 2021-02-04 | 2021-06-08 | 杭州海世嘉生物科技有限公司 | Non-gynecological exfoliated cell preservation solution, kit containing preservation solution and application |
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