CN102866052A - Cell staining kit and use method thereof - Google Patents
Cell staining kit and use method thereof Download PDFInfo
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- CN102866052A CN102866052A CN201210392692XA CN201210392692A CN102866052A CN 102866052 A CN102866052 A CN 102866052A CN 201210392692X A CN201210392692X A CN 201210392692XA CN 201210392692 A CN201210392692 A CN 201210392692A CN 102866052 A CN102866052 A CN 102866052A
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Abstract
The invention provides a cell staining kit and a use method thereof. The kit is characterized by comprising a fig leaf extract, a basic dye, an acid dye and a nucleus staining reagent. The fig leaf extract is used for conducting early diagnosis for cervical carcinoma for the first time, the affinity of cells for the staining reagent is improved, the staining efficiency and the cell discrimination rate are improved, and the accuracy of cervical carcinoma diagnosis is improved.
Description
Technical field
The present invention relates to a kind of cell staining reagent box and using method thereof, refer in particular to a kind of cell staining reagent box and using method thereof for cervical carcinoma early diagnosis and discriminating.
Background technology
Cervical carcinoma is one of common cancer that affects WomanHealth, and its incidence of disease is only second to breast cancer and occupies the second of female tumor.The whole world has 500,000 new cases of cervical cancer every year approximately, and the Asia is about 380,000, about 150,000 examples of China, and annual about 230,000 women in the whole world die from cervical carcinoma, and the Asia is about 190,000, China about 80,000.These data show that cervical carcinoma has become grave danger (Ng E etc., CMAL, 2004,170 (10): 1545-1549 of Chinese women's health; Qiao Youlin, 2006,26 (12): 1293-1295).95% is squamous cell carcinoma (Squmous Cells Carcinoma, SCC) in the cervical carcinoma, and the cure rate of clinical first phase can reach 80～90%, is 60～70% during the second phase, and three phases that entered are 40～50%, only have 10% cure rate but develop into the fourth phase.Therefore precancerous lesions of uterine cervix is comprised that cervical intraepithelial neoplasia becomes (Cervical Intraepithelium Neoplasia, CIN) control and early diagnosis cervical carcinoma are key (the Huth WK of control its generation, development and prognosis, J Natl ComprCanc Netw, 2010,8:1329-1330), the method for detecting specificity of further exploring for the cervical carcinoma early diagnosis just seems particularly important.
Because the normal scabies secondary infection of cervical carcinoma is with obvious inflammatory reaction, at H﹠amp; Cancer cell often is submerged in almost illegible in the inflammatory cell in the E dyeing, or H﹠amp; Cancer cell matter is red in the E dyeing, examine to be blueness, and red blood cell often is red that inflammatory cell is blue, makes to seek comparatively difficulty of cancer cell, fails to pinpoint a disease in diagnosis easily.
Summary of the invention
The purpose of this invention is to provide a kind of cell staining reagent box and using method thereof, to strengthen cell to the compatibility of staining reagent, improve the rate of distinguishing of dyeing efficient and cell, improve the accuracy rate of diagnosis of cervical cancer.
In order to achieve the above object, the invention provides a kind of cell staining reagent box, it is characterized in that, comprise common fig leaf juice extract, basic-dyeable fibre, acid dyes and nuclei dyeing color reagent.
Preferably, described basic-dyeable fibre is the New Fuchsine dyeing liquor.
Preferably, described acid dyes is the BG dyeing liquor.
Preferably, described nuclei dyeing color reagent is the agent of Harris haematoxylin dyeing.
The present invention also provides the using method of above-mentioned cell staining reagent box, it is characterized in that, in turn includes the following steps:
The first step: after cell sample is fixing with fixing agent, wash with deionized water;
Second step: dye with the nucleus of nuclei dyeing color reagent to the cell sample of first step gained, after the tap water flushing, wash with deionized water again;
The 3rd step: hatch the cell sample of second step gained with the common fig leaf juice extract, wash with deionized water;
The 4th the step: with basic-dyeable fibre hatch the 3rd the step gained cell sample, wash with deionized water;
The 5th the step: with acid dyes hatch the 4th the step gained cell sample, wash with deionized water;
The 6th step: after the cell sample drying with the 5th step gained, mounting is observed.
Preferably, the concrete steps of the described first step are: be fixing 60min in 10% the trichloroacetic acid solution with cell sample in mass concentration, wash 10sec with deionized water.
More preferably, described cell sample is cell smear or histotomy.Wherein cell smear can be that the manual smear of cell or liquid basement membrane formula thin-layer cell are learned smear, and histotomy can be frozen section or paraffin section.
Preferably, the concrete steps of described second step are: to the nucleus of the cell sample of the first step gained 9min that dyes, behind tap water flushing 1min, wash 30sec with deionized water with the Harris haematoxylin, immerse deionized water 10sec again.
Preferably, the concrete steps in described the 3rd step are: hatch the cell sample 3min of second step gained with the common fig leaf juice extract, wash 1min with deionized water, immerse deionized water 10sec again.
Preferably, the concrete steps in described the 4th step are: hatch the 3rd cell sample 1min that goes on foot gained with the New Fuchsine dyeing liquor, wash 1min with deionized water, immerse deionized water 10sec again.
Preferably, the concrete steps in described the 5th step are: hatch the 4th cell sample 1min that goes on foot gained with the BG dyeing liquor, wash 30sec with deionized water, immerse deionized water 10sec again.
Preferably, the concrete steps in described the 6th step are: the cell sample of the 5th step gained is dried, use the neutral gum mounting.
The present invention uses fig leaf extract to be used for the cervical carcinoma early diagnosis first, to strengthen cell to the compatibility of staining reagent, improves the rate of distinguishing of dyeing efficient and cell, improves the accuracy rate of diagnosis of cervical cancer.
The present invention can increase the different colours discriminating on the basis that keeps form to cell, and this technology has the dual-use function of colouring discrimination and cellular morphology analysis.
The reaction velocity of the present invention dyeing is fast, and operation steps is simple, and weak point consuming time can satisfy and screen the required technical requirement of atypical cell simultaneously, for cervical carcinoma early diagnosis and antidiastole provide effective instrument.
Description of drawings
Fig. 1 be the inventive method dyeing normal/atrophy or change epithelial cell and the canal of uterine cervix epithelial cell microphoto of giving birth to.
Figure 1A is normal squamous cell, atrophy or changes the scaly epithelium of giving birth to: kytoplasm dyeing is most to be blue/green, nuclear staining: blue/green or pale red/pink colour, wherein the self-dissolving of atrophy squamous cell can cause the living scaly epithelium nuclear great majority of bare nucleus and change to be the rouge and powder look.The dyeing of a few cell kytoplasm is pale red/pink colour, is defined as normal surperficial squamous cell this moment by morphological criteria) (a is manual smear, and b is the TCT smear); Figure 1B is that agglomerating cervical squamous cells (more than 5 cells) most peripheral cells kytoplasms and karyon are green or blue, and cell mass centrocyte kytoplasm and karyon are pink or red (a is manual smear, and b is the TCT smear); Fig. 1 C is canal of uterine cervix epithelial cell, karyon and kytoplasm dyeing: red (a is manual smear, and b is the TCT smear).
Fig. 2 is the cell that is not true to type (CIN and SCC) dyeing microphoto.
Fig. 2 A is that the CIN1-2 nucleus increases, and nuclear chromatin is evenly distributed and is the coarse particle shape, or intensive engrain.Karyon dyeing takes on a red color, kytoplasm dyeing take on a red color (a is manual smear, and b is the TCT smear); Fig. 2 B is be not true to type cell and cancer cell of CIN3.
CIN3 cell or SCC cellular morphology are irregular, show as fine and smooth or coarse (graininess) nuclear of nuclear chromatin and are significant nuclear and split or groove.The SCC cell is less than CIN3 cell, and nucleus is coarse bulk, the remarkable irregular distribution of chromatin, and nuclear atypia is obvious, large kernel or bare nucleus often occur.CIN3 cell or SCC cell karyon and kytoplasm dyeing take on a red color.Often show hemorrhage (a is manual smear, and b is the TCT smear) with more red blood cell (red blood cell dyeing is green) in the cell background.
Fig. 3 is inflammatory cell and red blood cell dyeing microphoto.
Fig. 3 A is inflammatory cell: examine and be redness, kytoplasm dyeing is green/blue; Fig. 3 B is that erythrocyte kytoplasm all is green (a is manual smear, and b is the TCT smear).
The manual smear of Fig. 4 is the inventive method dyeing microphoto after difference is fixing.
The normal scaly epithelium of Fig. 4 A is blue/green (a:Spray
Spraying is fixing; B ethanol is fixed); Fig. 4 B; The cell that is not true to type is red (a:Spray
Spraying is fixing; B ethanol is fixed); Fig. 4 C; Cancer cell (the a:Spray that takes on a red color
Spraying is fixing; B ethanol is fixed).
Fig. 5 is that CIN and SCC histotomy dye and H﹠amp through the inventive method; (wherein A1-A4 is H﹠amp to the as a result microphoto of E dyeing; Four sections of E dyeing, B1-B4 is four sections of the inventive method dyeing).
Embodiment
For the present invention is become apparent, hereby with preferred embodiment, and cooperate accompanying drawing to be described in detail below.
Embodiment
One, the preparation method of kit and reagent:
A kind of cell staining reagent box, its box body is in-built fig leaf extract, basic-dyeable fibre, acid dyes and nuclei dyeing color reagent.
Described common fig leaf juice extract is to prepare in 70% the ethanolic solution to obtain by fig leaf extract (10: 1) (available from Xi'an Si Nuote Bioisystech Co., Ltd) being dissolved in volumetric concentration, and wherein the final concentration of fig leaf extract is 30% (w/v).
Described basic-dyeable fibre is New Fuchsine dyeing liquor (New Fuchsin solution), be to prepare in 20% the alcoholic solution to obtain by New Fuchsine powder (available from U.S. Sigma company) being dissolved in volumetric concentration, wherein the final concentration of New Fuchsine is 0.5% (w/v), leaves standstill lucifuge 24h before the use.
Described acid dyes is BG dyeing liquor (Light Green solution), be to prepare in 20% the alcoholic solution to obtain by BG powder (available from U.S. Merck company) being dissolved in volumetric concentration, the final concentration of BG is 0.4% (w/v), leaves standstill lucifuge 24h before the use.
Described nuclei dyeing color reagent is the agent of Harris haematoxylin dyeing, adopts the conventional method preparation.
Other reagent comprises 10% trichloroacetic acid solution (Trichloroacetic acid, TCA) and ethanolic solution, all adopts the conventional method preparation.
Two, case source:
Case 600 routine vaginal smears are respectively from attached tumour hospital of Nantong University, First People's Hospital, city of Kunshan, the People's Hospital, Rudong County, hydrangea garden, Nantong City community apricot woods gynaecological disease clinic, manual smear 440 examples wherein, TCT smear 160 examples.Respectively manual smear and TCT smear are dyeed and H﹠amp with kit of the present invention; E dyeing is made a definite diagnosis uterine neck SCC and the further open surgical biopsy of intraepithelial neoplasia CIN3 case of smear diagnosis.
Three, dyeing:
Step 1, smear are prepared:
(1) manual smear 440 examples, every example is coated with 4, and wherein 2 are adopted the spraying cell to fix (Spray
), respectively with H﹠amp in the inventive method dyeing and the step 3 in the step 2; E dyeing, other 2 to adopt volumetric concentrations be that 95% ethanolic solution is fixed, respectively with H﹠amp in the inventive method dyeing and the step 3 in the step 2; E dyeing.The spraying cell is fixed (Surgipath Medical Industries, INC.P.O.BOX 528/5205Rte.12 Richmond, IL60071) finish in the 2-5sec at manual smear, evenly be sprayed onto from left to right whole cellular regions apart from smear 30cm, repeat 3 times, spraying fixedly 10min is dried naturally.Ethanol is fixed: after manual smear is finished slide immersed volumetric concentration and be more than 95% the ethanolic solution 10min, naturally dry.
(2) TCT smear 160 examples, Uterine neck bush brushes that to immerse volumetric concentration behind the cell be more than 95% the ethanolic solution 10min, treat that cell centrifugation is by TCT instrument (ZP-b membrane type, Hubei Xiaogan Hongxiang Biological ﹠ Medical Equipment Technology Co., Ltd.) 2 TCT smears of every example preparation are respectively with H﹠amp in the inventive method dyeing and the step 3 in the step 2; E dyeing.
Step 2, the inventive method dyeing following (manual smear is identical with TCT smear step):
(1) is fixing 60min in 10% the trichloroacetic acid solution with cell smear in mass concentration, washs 10sec with deionized water.
(2) with the Harris haematoxylin to the nucleus 9min that dyes, behind tap water flushing 1min, wash 30sec with deionized water, immerse again deionized water 10sec.
(3) hatch 3min with the common fig leaf juice extract, wash 1min with deionized water, immerse again deionized water 10sec.
(4) hatch 1min with the New Fuchsine dyeing liquor, wash 1min with deionized water, immerse again deionized water 10sec.
(5) hatch 1min with the BG dyeing liquor, wash 30sec with deionized water, immerse again deionized water 10sec.
(6) dry, use the neutral gum mounting, deposit, observe thereafter for 4 ℃ after dyeing is finished.
Step 3, H﹠amp; E dyeing: manual smear routinely method of operating is used Yihong coloring agent staining cell matter respectively with Harris haematoxylin dyeing agent staining cell nuclear.The programming automation control dyeing that the TCT smear designs in advance by liquid base instrument.
Step 4, tissue section strain: to uterine neck SCC and the further open surgical biopsy of intraepithelial neoplasia CIN3 case of smear diagnosis, sample through mass concentration be that 4% formalin is fixed, paraffin embedding, 4 μ m are thick in section, be used in the method identical with step 2 and step 3 and carry out respectively the inventive method dyeing and H﹠amp; Pathological diagnosis is carried out in E dyeing.
Four, observe:
Examine the inventive method stain smear under 10 times of mirrors, on average each visual field has 8 cells at least.20 times and 40 times of any suspicious or cluster forming cells of Microscopic observation, special concern is red dyes or purple transfect cell.
Carry out 95% credibility interval (Confidence Intervals, CI) appraisal for several inspections, adopt formula: susceptibility=true positives case/(true positives case+false negative case) * 100%; Specificity=true negative case/(true negative case+false positive case) * 100%.
Five, result:
1, the form of the inventive method dyeing vaginal cell and color performance:
The inventive method dyeing vaginal cell has shown the morphological feature of cell preferably in manual smear and TCT smear, and cell outline is clear, cell membrane complete (sometimes can occur＜20% cell lost membrane integrity, overflowed but have no kytoplasm).The color of simultaneously various types of cells demonstration is closely related with form.In manual smear 440 examples, be diagnosed as uterine neck SCC 14 examples, CIN58 example (CIN3:21 example, CIN1-2:37 example), inflammation change (comprising mould, trichomonas infection etc.) 328 example and relative normal cell 40 examples.Uterine neck SCC 18 examples, CIN 58 examples (CIN3:23 example, CIN1-2:35 example), inflammation change (comprising mould, trichomonas infection) 75 examples and relative normal cell 9 examples in TCT smear 160 examples.
(1) normal epithelium cell
Shallow table squamous cell comprises basal cell, intermediate cell and mature cell.Cell shape from the basal cell to the mature cell is from the ellipse to the polygon, the cell monokaryon from big to small, tenuigenin is from less to more.Normal single squamous cell (non-agglomerating cell) kytoplasm is blue or green after the inventive method dyeing.The atrophy squamous cell is 5 times of intermediate cell normally.Self-dissolving may cause bare nucleus.Nucleus is the rouge and powder look after the inventive method dyeing, and the tenuigenin overwhelming majority is blue or green.It is oval to change the squamous cell of giving birth to, and the nucleus great majority are the rouge and powder look after the inventive method dyeing, and tenuigenin is blue/green or rouge and powder look.In agglomerating Cervix Squamous Cell (more than 5 cells), most peripheral cells kytoplasms and karyon are green or blue, and the cell mass centrocyte is pink or red.Canal of uterine cervix epithelial cell cube, the nuclear circle is less, and kytoplasm is few, the agglomerating arrangement of cell.Kytoplasm and nuclear are cerise (as shown in Figure 1) after the inventive method dyeing.
(2) be not true to type cell and cancer cell
The cell that is not true to type often appears in the focus of hyperphlogosis or CIN, and the latter is further divided into low level CIN1-2 and high-level CIN3.The principal feature of cell be not true to type in the inflammation for nuclear increases, be about 2.5～3 times of intermediate cell nuclear, but nuclear chromatin or nuclear morphology rule.The CIN1-2 nucleus increases to more than 3 times of intermediate cell nuclear, and nuclear chromatin is evenly distributed and is the coarse particle shape, or intensive engrain, often without kernel or not remarkable.The CIN3 cell size differs, examines abnormity, and common significant nuclear splits or groove, and nuclear chromatin can fine and smooth or coarse (graininess), and kernel lacks as be or accidental.SCC cell and CIN3 feature similarity, but nuclear atypia is more obvious, large kernel or bare nucleus often occur, nuclei dyeing chromaticness skewness, coarse bulk.Normal with more red blood cell in the background of cancer cell, hemorrhage by due to the cancer cell invasion and attack of prompting.Be not true to type after the inventive method dyeing cell and cancer cell kytoplasm and karyon all takes on a red color (as shown in Figure 2)
(3) inflammatory cell and red blood cell
The less lobulated of inflammatory cell nuclear, the few or disappearance of tenuigenin, karyon takes on a red color (sometimes can be blue or green＜20%), and the inflammatory cell kytoplasm is blue or green.Erythrocyte kytoplasm all is green (as shown in Figure 3).
The dyeing of 2 the inventive method and H﹠amp; The comparison of E dyeing:
(1) in manual smear and TCT smear, compares respectively the inventive method dyeing and H﹠amp; The diagnostic result (table 1) of E dyeing
The dyeing of table 1 the inventive method and H﹠amp; E dyes relatively
2 routine SCC are arranged at H﹠amp in the inventive method inspection; Be diagnosed as CIN3 in the smear of E dyeing, 1 routine CIN3 arranged at H﹠amp; Be diagnosed as CIN2 in the smear of E dyeing.The inventive method check result and TCT plate coating checking come to the same thing.
3 manual smears are the inventive method coloration result after difference is fixing
No matter be that the spraying cell is fixed or 95% ethanol is fixed, smear is through the equal morphological feature of showed cell preferably of the inventive method dyeing, the normal scaly epithelium kytoplasm dyeing overwhelming majority is blue/green, nuclear staining is blue/green or pale red/pink colour, canal of uterine cervix epithelial cell kytoplasm and nuclear staining are cerise, and cell and cancer cell karyon and the matter of not being true to type dyeing takes on a red color.Inflammatory cell nuclear is for red, and kytoplasm dyeing great majority are green/blue, and it is green that red blood cell all is.The color that cell shows is closely related with form.Result's (Fig. 4, table 2) in full accord of different fixing middle side's dyeing of the present invention.
The result of the different fixing middle the inventive method dyeing of table 2
4CIN 3 and SCC pathological biopsy are through the inventive method dyeing and H﹠amp; The result of E dyeing
To uterine neck SCC 33 examples (manual smear 14 examples, TCT smear 19 examples) and the further open surgical biopsy of CIN3 43 examples (manual smear 21 examples, TCT smear 22) of the inventive method Staining for Diagnosis, pathological section carries out respectively the inventive method dyeing and H﹠amp; E dyeing.The inventive method coloration result shows that normal scaly epithelium and cuticular layer are green/blue, the reaction that takes on a red color of CIN and SCC zone, the reaction (as shown in Figure 5) that also takes on a red color of neck tube body of gland.
Result according to the biopsy histology diagnosis estimates respectively the inventive method and H﹠amp in manual smear and the TCT smear; Susceptibility and the specificity (such as table 3,4) of E dyeing.
The inventive method and H﹠amp in the more manual smear of table 3; Susceptibility and the specificity of E dyeing
Table 4 compares the inventive method and H﹠amp in the TCT smear; Susceptibility and the specificity of E dyeing
Claims (12)
1. a cell staining reagent box is characterized in that, comprises common fig leaf juice extract, basic-dyeable fibre, acid dyes and nuclei dyeing color reagent.
2. cell staining reagent box as claimed in claim 1 is characterized in that, described basic-dyeable fibre is the New Fuchsine dyeing liquor.
3. cell staining reagent box as claimed in claim 1 is characterized in that, described acid dyes is the BG dyeing liquor.
4. cell staining reagent box as claimed in claim 1 is characterized in that, described nuclei dyeing color reagent is the agent of Harris haematoxylin dyeing.
5. the using method of each described cell staining reagent box among the claim 1-4 is characterized in that, in turn includes the following steps:
The first step: after cell sample is fixing with fixing agent, wash with deionized water;
Second step: dye with the nucleus of nuclei dyeing color reagent to the cell sample of first step gained, after the tap water flushing, wash with deionized water again;
The 3rd step: hatch the cell sample of second step gained with the common fig leaf juice extract, wash with deionized water;
The 4th the step: with basic-dyeable fibre hatch the 3rd the step gained cell sample, wash with deionized water;
The 5th the step: with acid dyes hatch the 4th the step gained cell sample, wash with deionized water;
The 6th step: after the cell sample drying with the 5th step gained, mounting is observed.
6. the using method of cell staining reagent box as claimed in claim 5 is characterized in that, the concrete steps of the described first step are: be fixing 60min in 10% the trichloroacetic acid solution with cell sample in mass concentration, wash 10sec with deionized water.
7. the using method of cell staining reagent box as claimed in claim 5 is characterized in that, described cell sample is cell smear or histotomy.
8. the using method of cell staining reagent box as claimed in claim 5, it is characterized in that, the concrete steps of described second step are: with the Harris haematoxylin to the nucleus of the cell sample of the first step gained 9min that dyes, behind tap water flushing 1min, wash 30sec with deionized water, immerse again deionized water 10sec.
9. the using method of cell staining reagent box as claimed in claim 5, it is characterized in that, the concrete steps in described the 3rd step are: hatch the cell sample 3min of second step gained with the common fig leaf juice extract, wash 1min with deionized water, immerse deionized water 10sec again.
10. the using method of cell staining reagent box as claimed in claim 5, it is characterized in that, the concrete steps in described the 4th step are: hatch the 3rd cell sample 1min that goes on foot gained with the New Fuchsine dyeing liquor, wash 1min with deionized water, immerse deionized water 10sec again.
11. the using method of cell staining reagent box as claimed in claim 5, it is characterized in that, the concrete steps in described the 5th step are: hatch the 4th cell sample 1min that goes on foot gained with the BG dyeing liquor, wash 30sec with deionized water, immerse deionized water 10sec again.
12. the using method of cell staining reagent box as claimed in claim 5 is characterized in that, the concrete steps in described the 6th step are: the cell sample of the 5th step gained is dried, use the neutral gum mounting.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109668771A (en) * | 2019-02-26 | 2019-04-23 | 长沙协大生物科技有限公司 | A kind of liquid-based cast-off cells dyeing liquor and its colouring method |
| CN111380729A (en) * | 2018-12-27 | 2020-07-07 | 上海细胞治疗集团有限公司 | Preparation method and application of cell smear |
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| CN1920559A (en) * | 2005-08-24 | 2007-02-28 | 赵翀 | Cellular biological technique, reagent kits and preparation device |
| CN101680895A (en) * | 2007-02-06 | 2010-03-24 | J制药股份有限公司 | Kit for deciding degree of malignancy in prostate cancer and method of using the same |
| CN102268409A (en) * | 2011-06-28 | 2011-12-07 | 南方医科大学 | Monoclonal antibody against human protein RN181 and hybridoma cell strain 3952 and kit |
| WO2012070041A1 (en) * | 2010-11-22 | 2012-05-31 | Zetiq Technologies Ltd. | Methods and kits for differential staining of abnormal urinary system cells |
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| CN1421460A (en) * | 2002-12-03 | 2003-06-04 | 中国医学科学院医药生物技术研究所 | Conjugate of lidamycin, monoclonal antibody and its Fab'segment and its application in targeting treatment of colon cancer and other tumors |
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| CN101680895A (en) * | 2007-02-06 | 2010-03-24 | J制药股份有限公司 | Kit for deciding degree of malignancy in prostate cancer and method of using the same |
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| CN111380729A (en) * | 2018-12-27 | 2020-07-07 | 上海细胞治疗集团有限公司 | Preparation method and application of cell smear |
| CN109668771A (en) * | 2019-02-26 | 2019-04-23 | 长沙协大生物科技有限公司 | A kind of liquid-based cast-off cells dyeing liquor and its colouring method |
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Application publication date: 20130109 |