CN102260731B - Enzyme-labeling-liquid-based cytology staining kit for screening bladder cancer - Google Patents
Enzyme-labeling-liquid-based cytology staining kit for screening bladder cancer Download PDFInfo
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Abstract
The invention relates to application of an enzyme-labeling-liquid-based cytology technology to the screening of bladder cancer. In the technology, a cell sample which is tabletted by a liquid-based tabletting method is treated by an acid phosphatase chemical staining method, and a bright red precipitate is formed in cytoplasm of abnormal cells by specific staining to ensure that the abnormal cells have remarkable marks. The invention provides a kit for the detection. The kit mainly comprises an enzyme-labeling-liquid-based cytology staining reagent, enzyme-labeling-liquid-based cytology preserving fluid and settlement buffer solution, and can provide high detection specificity and a good staining effect.
Description
Technical field
The present invention relates to detection kit and the application thereof of bladder cancer, more precisely utilize enzyme mark liquid basal cell to learn a skill examination tumor of bladder.This technology utilizes the cell sample of acid phosphatase chemical coloring process to the film-making of liquid base method of tableting to process, and utilizes this specific stain to make to form distinct red precipitate in abnormal cells endochylema, make abnormal cells with the technology of visible marking.
Background technology
Bladder cancer is the modal urological cancer of China, and the bladder cancer of about 90% is Urothelial Carcinoma of Bladder.The recurrence rate of bladder cancer is very high, and the patient of 60% ~ 70% may be recurred, and the patient of 11% recurrence can be in progress as invasive tumor.At present for Diagnosis of Bladder and follow up a case by regular visits to and depend on cystoscopy and urinary cytology inspection.The former is the gold standard of diagnosing bladder cancer, but is invasive inspection, and patient compliance is poor, and price comparison is expensive, should not as routine inspection; Although the latter's specific degree is high and without wound, tumor sensitivity minuent being shown to shallow property is low, easily produce false-negative result.Therefore, find susceptibility and the high bladder cancer tumor markers of specific degree and receive increasing concern as the early diagnosis of bladder cancer, monitoring and prognosis evaluation.
The sampling method of urinary cytology mainly contains nature and stays urine and Intravesical instillation two kinds, and the middle portion usually getting midstream urine or G Solution is originated as cytologic specimen.Badalamen etc. point out that the sensitivity of Intravesical instillation sample cytolgical examination is 61%, and naturally stay the sensitivity of urine to be 41%; Urine three sensitivity is repeatedly stayed to be 60%, still lower than the cytolgical examination of single Intravesical instillation.
Urinary cytology film-making analytical procedure mainly contains Conventional slide and the liquid basal cell recently carried out learns a skill, easily disturbed by red corpuscle during Conventional slide read tablet, and the liquid basal cell impurity can removing interference diagnosis that learns a skill makes the liquid basal cell sheet of clear background, preserve cytological appearance better, make observation more easy.Liquid based cytology comprises ThinPrep cytology test and liquid base sedimentation cytologic technology, the cast-off cells of collection are transferred in the special bottle containing conserving liquid by the former, filtered cell and magazins' layout by filtering membrane, finally on slide, make the cell thin that diameter is 20mm, row pap staining after fixing, once can only process a sample.The latter be by cast-off cells and conserving liquid centrifugal by specific gravity liquid after, with the method for natural sedimentation by the suspicious cells in sample and magazins' layout, on slide, finally make the cell thin that diameter is 17-20mm, many parts of samples can be processed simultaneously.Enzyme mark liquid based cytology is mainly used in the examination of tumor of bladder.This technology utilizes the cell sample of acid phosphatase chemical coloring process to the film-making of liquid base method of tableting to process, and utilizes this specific stain to make to form distinct red precipitate in abnormal cells endochylema, make abnormal cells with the technology of visible marking.Enzyme staining is used for the diagnosis of clinical molecular level by this technology, has susceptibility high, the features such as false negative rate is low.The utilization of this technology can help doctor to find abnormal cells rapidly.
Acid phosphatase (Acid Phosphatase, ACP) is one of lysosomal marker enzyme, its active reaction activation situation of Cytolysosome.In bladder cancerous tumor cell, acid phosphatase expressed in abundance, then can't detect it and expresses in normal cell.It is utilize the feature that in abnormal epithelial cell, ACP activity increases that enzyme mark liquid basal cell learns a skill, and screens normal and sick cell by detecting ACP activity.
HE dyeing process current is at present Thinprep pap test method of tableting and Conventional slide, and aforesaid method relies on merely doctor to provide check result according to cellular form and karyomorphism, and workload is large, and subjective, false negative rate is high.The invention reside in and provide a kind of method utilizing the cell sample of acid phosphatase chemical coloring process to the film-making of liquid base method of tableting to carry out processing examination bladder cancer, the utilization of this technology can increase the visuality of observation greatly, improves the accuracy of diagnosis.And it is simple to operate, consuming time short, highly sensitive.
Summary of the invention
The present invention with the specific reaction of acid phosphatase enzyme catalysis generate product and azo and pay red precipitate that pinkish red coupling generates for marking, from molecular biology level qualification abnormal cells, ensure that accuracy and the sensitivity of diagnosis.Enough help doctor to find sick cell rapidly, doctor only need carry out karyotyping for staining positive cells when diagnosing simultaneously, reduces working doctor amount.
In conjunction with liquid-based cell sample manufacturing technology and conventional nuclear staining, not only remain cellular form to the full extent, also make abnormal cells have the red dye of endochylema and core increase, the feature of nuclear hyperchromatism simultaneously, make the form of cell caryoplasm abnormal proportion more clear, cellular informatics is maximized, is easier to interpretation.
Enzyme mark liquid basal cell learns a skill and utilizes that in abnormal bladder transitional epithelium, have highly active Phosphoric acid esterase be that specific mark is developed, its central principle utilizes naphtholate as the substrate of acid phosphatase, and the diazonium salt utilizing azo-compound and nitrate effect to be formed forms red precipitate as conjugate in abnormal cells.
Nearest cellular elements experimental studies results prompting, in tumour cell, p53 significantly reduces relative to normal cell or shows as the sudden change losing normal function, removes p53 like this to the suppression of cell autophagy degeneration system, thus strengthens the activity of this system.As can be seen here, tumour cell may be able to be shown by lysosomal activity.
Acid phosphatase is one of lysosomal marker enzyme, can be reflected the activation situation of lysosomal enzyme by the activity of detection of acidic Phosphoric acid esterase.Infer consistent with us, in normal transitional epithelial cell, acid phosphatase is negative, and abnormal epithelial cell acid phosphatase is positive.And, now, do not relate to the correlative study that in urine sample cancer cells acid phosphatase high expression level detects, more not by research that acid phosphatase high expression level in urine sample is associated with bladder cancer yet.The present invention's research shows, only have urological cancer (especially bladder cancer) high expression level of acid phosphatase in abnormal transitional epithelial cell just can be detected in urine sample, namely in urine sample, the high expression level of this enzyme is special to bladder cancer.It is according to the bladder abnormal cells examination technology of acid phosphatase in the feature development research of abnormal epithelial cell specificity overexpression that enzyme mark liquid basal cell learns a skill.Clinical study shows, compared with traditional HE dyeing process, this technology has fast and the feature of high sensitivity, and diagnosis and generaI investigation for the abnormal transitional epithelial cell of bladder provide a convenient and reliable method.
The present invention relates to a kind of detection kit and application thereof of bladder cancer, described test kit comprises enzyme mark liquid based cytology staining reagent, enzyme mark liquid based cytology conserving liquid and Sedimentation Buffer.
Staining reagent comprises by stationary liquid, staining fluid, hydrochloric acid magenta, Sodium Nitrite, sodium acetate soln, Hematorylin redyes liquid and filter paper forms.More preferably by
(1) stationary liquid: 10% formalin;
(2) enzyme mark liquid based cytology staining fluid: 2-30.5g/l naphthols AS-BI phosphoric acid salt is dissolved in dimethyl formamide (i.e. the phosphatic dimethyl formamide solution of naphthols AS-BI);
(3) hydrochloric acid is pinkish red: 1-10% hydrochloric acid is pinkish red;
(4) Sodium Nitrite: 0.1-0.5M NaNO
2;
(5) sodium acetate soln: 1.5-4.5M, pH 4.5-5.9;
(6) Hematorylin redyes liquid;
(7) filter paper composition.
Enzyme mark liquid based cytology conserving liquid of the present invention has anticorrosion, removing impurity, lysed erythrocyte function, uses that it coordinates Sedimentation Buffer of the present invention to carry out described film-making, the effect of dyeing is better than traditional conserving liquid and Sedimentation Buffer.Shown in conserving liquid of the present invention is composed as follows:
(1) 45% methyl alcohol
(2)0.2-0.5mM EDTA
(3)0.1mM NaCl
(4) 0.1M acetate buffer solution, pH 4-6
(5)dd H
2O
Sedimentation Buffer of the present invention is better than conventional buffer equally, and the image obtained after its film-making, dyeing is more clear, is convenient to observe.Sedimentation Buffer of the present invention has following two kinds of formulas:
Formula a:
0.5XPBS，pH＝7.0-7.4，2-5mM EDTA；
Formula b:
10mM Tris-HCl pH＝7.0-7.4，2-5mM EDTA，0.9％(m/v)NaCl。
The invention still further relates to the purposes of described enzyme mark liquid based cytology staining reagent in the test kit for the preparation of sieving and diagnosis bladder cancer patients.
The invention still further relates to described enzyme mark liquid based cytology staining reagent and the purposes of conserving liquid in the test kit for the preparation of sieving and diagnosis bladder cancer patients.
In addition, the invention still further relates to described enzyme mark liquid based cytology staining reagent, conserving liquid and the purposes of Sedimentation Buffer in the test kit for the preparation of sieving and diagnosis bladder cancer patients.
Accompanying drawing explanation
Fig. 1: enzyme mark liquid based cytology technology tradition method film-making result photo (200X), the cell incarnadined is bladder abnormal cells, and proved by pathology is intraepithelial neoplasia (cin), and namely pathology is early stage.In figure
instruction red corpuscle, and
instruction abnormal cells group.
Fig. 2: enzyme mark liquid based cytology technology tradition method film-making result photo, wherein left figure is 200X; Right figure is 400X; In figure, blue plate-like small-particle is red corpuscle.In figure
indicate normal Urothelial cell,
instruction abnormal cells.
Fig. 3: liquid basal cell sedimentation film-making coloration result: the picture left above is 200X.Lower-left figure is abnormal cells enlarged view in the picture left above, the red dye of visible kytoplasm, prompting ACP high expression level; Top right plot is normal epithelial cell enlarged view in the picture left above, incarnadines without kytoplasm; Bottom-right graph is the paracytic enlarged view of lens sections sample core in the picture left above, can see the red dye of kytoplasm equally.
Fig. 4: positive control: bladder cancer cell line BIU-87 (purchased from China typical culture collection center, Wuhan) enzyme mark liquid based cytology dyeing (400X).In visual fields, cancer cells all has red dye in various degree, and red dye increases with nuclear atypia degree and deepens.
Fig. 5: bladder cancer cell line BIU-87 is preserved the coloration result after 7 days respectively in enzyme mark liquid based cytology conserving liquid of the present invention and traditional conserving liquid.Wherein, Fig. 5 A is after BIU-87 preserves one week in conserving liquid, natural subsidence film-making in the Sedimentation Buffer of formula a, enzyme mark liquid based cytology coloration result (400x); Fig. 5 B is after BIU-87 preserves one week in conserving liquid of the present invention, natural subsidence film-making in Sedimentation Buffer described in formula b, enzyme mark liquid based cytology coloration result (400x); Fig. 5 C is after BIU-87 preserves one week in traditional conserving liquid, natural subsidence film-making in supporting damping fluid, enzyme mark liquid based cytology coloration result (400x).
Specific embodiment
Embodiment 1
One, enzyme mark liquid based cytology staining kit composition:
(1) stationary liquid: 10% formalin;
(2) enzyme mark liquid based cytology staining fluid: 2-30.5g/l naphthols AS-BI phosphoric acid salt is dissolved in dimethyl formamide;
(3) hydrochloric acid is pinkish red: 1-10% hydrochloric acid is pinkish red;
(4) Sodium Nitrite: 0.1-0.5M NaNO
2;
(5) sodium acetate soln: 1.5-4.5M, pH 4.5-5.9;
(6) Hematorylin redyes liquid;
(7) filter paper
Two, conserving liquid composition:
(1) 45% methyl alcohol;
(2)0.2-0.5mM EDTA；
(3)0.1mM NaCl；
(4) 0.1M acetate buffer solution, pH 4-6;
(5)dd H
2O。
Three, Sedimentation Buffer composition:
Formula a:
0.5XPBS，pH＝7.0-7.4，2-5mM EDTA；
Formula b:
10mM Tris-HCl pH＝7.0-7.4，2-5mM EDTA，0.9％(m/v)NaCl.
Three, sampling and operation
A. conventional film-making
Conventionally leave and take CCMS 15mL in early morning, the centrifugal 5min of 4000rpm, incline liquid.Adopt traditional ordinary method film-making: cell take out be placed between two slide glasss, pressure reversal is pulled open a little, two uniform smears of thickness.15mL urine sample can open smear by 2-4 processed.After cell drying, fix (reagent A) 1min with 10% formalin, dye according to staining procedure afterwards.
B. liquid base natural sedimentation film-making
Conventionally leave and take midstream urine 15mL in early morning, the centrifugal 5min of 4000rpm, incline liquid.Use conserving liquid re-suspended cell, in conserving liquid, cell can preserve one to surrounding.During film-making, conserving liquid is centrifugal, incline liquid, uses Sedimentation Buffer re-suspended cell.The film-making of natural subsidence method, (reagent A) 1min fixed by 10% formalin.Special conserving liquid has anticorrosion, removing impurity, lysed erythrocyte function.
Meanwhile, dye as positive control with bladder cancer cell line BIU-87 (purchased from China typical culture collection center, Wuhan) enzyme mark liquid based cytology.
Three, staining procedure:
1. by fully clean for the smear water after fixing
2. smear is put into enzyme mark liquid based cytology staining fluid and be placed in 25-37 DEG C of dyeing 20-30 minute
3. fully clean with water
4. smear is put into Hematorylin and redye liquid 5 minutes, running water 5 minutes
6. mounting, microscopy
Four, coloration result:
Fig. 1 and Fig. 2 is enzyme mark liquid based cytology technology tradition method film-making result photo; Fig. 3 and Fig. 4 is liquid basal cell sedimentation film-making coloration result.Can see by above-mentioned Fig. 1-3, abnormal epithelial cell kytoplasm is all incarnadined, and normal cell non-coloring phenomenon; And liquid basal cell sedimentation film-making coloration result is better than traditional method film-making, and result is more obvious, is more easy to observe.Meanwhile, in the dyeing of positive control bladder cancer cell line BIU-87 enzyme mark liquid based cytology, cancer cells all has red dye in various degree in visual fields, and red dye increases with nuclear atypia degree and deepens.
Embodiment 2
Choose and be accredited as each 3 examples of bladder cancer, liver cancer, lung cancer, large bowel cancer, cancer of the stomach, cervical cancer patient and Healthy People clinically, get urine sample according to the method for embodiment 1, and carry out the dyeing of enzyme mark liquid based cytology.Result shows, and 3 routine bladder cancer patients have all occurred that the kytoplasm in Fig. 1-3 incarnadines, and all incarnadine appearance without kytoplasm in all the other cancer patients's urine samples and Healthy People urine sample.
Above-mentioned test-results shows, detects that the high expression level of acid phosphatase has compared with high specific to bladder cancer in urine sample, and the method for the application and test kit can be used in sieving and diagnosis or auxiliary sieving and diagnosis bladder cancer patients.
The effect test of embodiment 3 enzyme mark liquid based cytology conserving liquid and Sedimentation Buffer
After bladder cancer cell line BIU-87 is preserved 7 respectively in the enzyme mark liquid based cytology conserving liquid described in the application and existing certain conserving liquid (purchased from Xiamen Maiwei Biotechnology Co., Ltd.), Sedimentation Buffer is coordinated to carry out film-making and dyeing, the result obtained as shown in Figure 5, the film-making carried out in enzyme mark liquid based cytology conserving liquid of the present invention and Sedimentation Buffer and Color (Fig. 5 A, 5B) successful are better than cellular control unit conserving liquid and Sedimentation Buffer (Xiamen Maiwei Biotechnology Co., Ltd., Fig. 5 C).Its concrete comparative result ("+" multilist shows that effect is more excellent) as shown in the table:
Result is visible thus, and enzyme mark liquid based cytology conserving liquid of the present invention and Sedimentation Buffer obviously will be better than compared with control cells conserving liquid and Sedimentation Buffer (stepping prestige biological).
Claims (1)
1. for a test kit for auxiliary sieving and diagnosis bladder cancer, it is characterized in that, described test kit is made up of enzyme mark liquid based cytology staining reagent, enzyme mark liquid based cytology conserving liquid and Sedimentation Buffer;
Wherein, described enzyme mark liquid based cytology staining reagent concrete composed as follows shown in:
(1) stationary liquid: 10% formalin,
(2) staining fluid: 2-30.5g/l naphthols AS-BI phosphoric acid salt is dissolved in dimethyl formamide,
(3) hydrochloric acid is pinkish red: 1-10% hydrochloric acid is pinkish red,
(4) Sodium Nitrite: 0.1-0.5M NaNO
2,
(5) sodium acetate soln: 1.5-4.5M, pH 4.5-5.9,
(6) Hematorylin redyes liquid,
(7) filter paper;
Described enzyme mark liquid based cytology conserving liquid concrete composed as follows shown in:
(1) 45% methyl alcohol,
(2)0.2-0.5mM EDTA，
(3)0.1mM NaCl，
(4) 0.1M acetate buffer solution, pH 4-6,
(5)dd H
2O；
Described Sedimentation Buffer has formula a and formula b two kinds, and its concrete composition is respectively:
Formula a:0.5XPBS, pH=7.0-7.4,2-5mM EDTA;
Formula b:10mM Tris-HCl pH=7.0-7.4,2-5mM EDTA, 0.9% (m/v) NaCl.
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| CN105424448A (en) * | 2015-11-06 | 2016-03-23 | 合肥锦慈康生物技术有限公司 | Acid phosphatase staining method for distinguishing malignant serosal cavity effusion mesothelial cells from cancer cells and application thereof |
| CN107271246B (en) * | 2017-07-11 | 2018-08-14 | 广州江元医疗科技有限公司 | A kind of immune p16INK4a antigens for Thinprep pap test preserve liquid and preparation method thereof |
| CN109490049A (en) * | 2018-12-27 | 2019-03-19 | 大连大学 | A kind of haematoxylin dyeing liquid and preparation method |
| CN111238889A (en) * | 2020-01-16 | 2020-06-05 | 江西业力医疗器械有限公司 | Liquid-based fungus detection method based on flaking method |
| CN111972400A (en) * | 2020-08-18 | 2020-11-24 | 武汉瑞新昌生物科技有限公司 | Treatment and preservation reagent for liquid-based cells and microorganisms |
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Address after: 230088 Anhui city of Hefei province high tech Zone Huayi Science Park E2-301 Patentee after: Hefei Jiusheng Biological Medicine Co Ltd Address before: 230088 Anhui city of Hefei province high tech Zone Huayi Science Park E2-301 Patentee before: Hefei Anyon Biopharmaceuticals Co., Ltd. |