CN103033409B - The histocyte colouring method improved and application thereof - Google Patents
The histocyte colouring method improved and application thereof Download PDFInfo
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Abstract
The disclosure relates to cervical squamous cells colouring method, the kit of improvement and is diagnosing the application in individual cervical squamous cells cancer method.Described method or kit comprise cellular acid phosphatase dyeing reagent and improvement pap staining reagent and application.The disclosure also relates to the kit of the individual cervical squamous cells cancer of diagnosis or precancerous lesion.
Description
Technical field
The application relates to histology and cytological staining methods and application, particularly cervical squamous cells colouring method and application thereof.
Background technology
Cervical carcinoma is one of common gynecologic malignant tumor, and the incidence of disease occupies the second of global female malignant, and often have 500,000 cervical carcinoma new cases every year, wherein the case of 80% occurs in developing country.There is new cases 13.15 ten thousand example every year in China, account for 28.8% of world's cervical carcinoma new cases sum.In recent years, studies have found that the young case load of cervical carcinoma has the trend increased year by year, age of onset is rejuvenation trend, and the generation of cervical carcinoma, development have a quantitative change, are gradient to the process of sudden change, therefore, early detection, early diagnosis and early stage treatment cervical carcinoma, can reduce its incidence of disease and case fatality rate.Examination is the Main Means of prevention and early diagnosis cervix cancer at present.
Since cell smear Papanicolaou's vaginal smear technique (Pap-test) birth of the George Papanicolaou forties in 20th century, the Pasteur's 5 grades of classification continuing 40 years make the incidence of disease of cervix cancer reduce 70%-80%, simultaneously because its cost-effectiveness is high very universal in developing country's application.But owing to being subject to many factors, false positive rate and the false negative rate of Papanicolaou's vaginal smear technique are too high.Invent a lot of new method and new technology in recent years, having comprised: Thinprep technology (Thin Prep, Liquid-BasedMonolayers TCT)-be intended to has improved the technology of slice-making quality; Fluorescence inspects the method for the luminous amplifying technique of method (Speculoscopy)-applied chemistry in conjunction with Pap; The technology of the DNA of hybrid capture 2 (Hybrid Capture 2) technology (HC2)-detection human papilloma virus (HPV); And detect p16INK4a albumen with immunocytochemical method (Immunocytochemical).However, Papanicolaou's vaginal smear technique is still the Main Means of current examination cervical carcinoma.
Summary of the invention
First aspect, relates to the cervical squamous cells colouring method of improvement herein, comprises
Acid phosphatase stain is carried out to described cervical squamous cells; And
Pap staining is carried out to described cervical squamous cells.
Second aspect, relates to the cervical squamous cells staining kit of improvement herein, comprises
The reagent of cellular acid phosphatase dyeing; And
Pap staining reagent.
The third aspect, relate to the method diagnosing individual cervical squamous cells cancer herein, described method comprises,
Collect individual cervical squamous cells sample,
Fixing described sample,
Acid phosphatase stain is carried out to described cervical squamous cells,
Pap staining is carried out to described cervical squamous cells, and
Judge whether cell is cancer cell or precancerous lesion by pathology professional and technical personnel according to colour developing result.
Fourth aspect, relate to the diagnostic kit of individual cervical squamous cells cancer herein, described kit comprises,
The reagent of fixing individual cervical squamous cells sample;
The reagent of cellular acid phosphatase dyeing;
Pap staining reagent, and
Optionally comprise the instructions instructing pathology professional and technical personnel to judge staining cell.
5th aspect, relates to special immobile liquid herein, and described immobile liquid comprises
A liquid: sodium citrate buffer solution; With
B liquid: acetone, formaldehyde hybrid working liquid.
Accompanying drawing explanation
Fig. 1, Fig. 2 are the Normal squamous epithelium (Fig. 1, Fig. 2 are respectively 20X and 40X) of traditional pap staining display.Different colors is presented because of the acidophilia of squamous cell and basophilous difference.
Fig. 3, Fig. 4 are the Normal squamous epithelium (Fig. 3, Fig. 4 are respectively 10X and 20X) of modified pap staining display.
Fig. 5, Fig. 6 are the Atypical squalors cell (ASC-US) that acid phosphatase stain combines the interrogatory of the new method dyeing display of the pap staining improved.
Fig. 7 is the new method dyeing display squamous cell abnormal in early stage that acid phosphatase stain combines the pap staining improved.
Fig. 8 is new method dyeing display dermoid cancer venereal disease change (SCC) that acid phosphatase stain combines the pap staining improved.
Fig. 9 shows the enzyme dyeing of Hela cell after traditional immobile liquid is fixed.
Figure 10 shows the enzyme dyeing of Hela cell after special immobile liquid is fixed.
Embodiment describes in detail
Inventor is surprised to find, and utilizes Cytochemical staining method to detect cervical cell acid phosphatase (CAP) active simultaneously in conjunction with the pap staining technology of improvement, can improve the Color of cervical squamous cells exception.
Accordingly, first aspect, relates to the cervical squamous cells colouring method of improvement herein, comprises
Acid phosphatase stain is carried out to described cervical squamous cells; And
Pap staining is carried out to described cervical squamous cells.
Second aspect, relates to the cervical squamous cells staining kit of improvement herein, comprises
The reagent of cellular acid phosphatase dyeing; And
Pap staining reagent.
The third aspect, relate to the method diagnosing individual cervical squamous cells cancer herein, described method comprises,
Collect individual cervical squamous cells sample;
Fixing described sample;
Acid phosphatase stain is carried out to described cervical squamous cells;
Pap staining is carried out to described cervical squamous cells; And
Judge whether cell is cancer cell or precancerous lesion by pathology professional and technical personnel according to colour developing result.
Fourth aspect, relate to the diagnostic kit of individual cervical squamous cells cancer herein, described kit comprises,
The reagent of fixed sample;
The reagent of cellular acid phosphatase dyeing;
Pap staining reagent, and
Optionally comprise the instructions instructing pathology professional and technical personnel to judge staining cell.
5th aspect, relates to special immobile liquid herein, and described immobile liquid comprises
A liquid: sodium citrate buffer solution; With
B liquid: acetone, formaldehyde hybrid working liquid.
In one embodiment, described cervical squamous cells comprises cervical squamous cells that is normal, abnormal, canceration and precancerous lesion thereof.In another embodiment, described abnormal, canceration or precancerous lesion cervical squamous cells comprise, Atypical squalors cell (ASC), it comprises the Atypical squalors cell (ASC-US) of (i) interrogatory, (ii) not except the Atypical squalors cell (ASC-H) of epithelium inner height pathology; Low Squamous cell lesions (LSIL); High-grade squamous epithelial lesion (HSIL); Squamous cell carcinoma (SCC).
Term used herein " individuality " refers to mammal, comprises people and other mammals.In this article, term " individuality " and " patient " can cross-reference, especially female mammal or female patients.
In one embodiment, three kinds of dyeing liquors, i.e. brazilwood extract dyeing liquid, orange G (OG) dyeing liquor and EA (Eosin Azure) dyeing liquor is contained for the pap staining pack in described method and kit.
In one embodiment, described brazilwood extract dyeing liquid can be that Harris brazilwood extract dyeing liquid or Gill improve brazilwood extract dyeing liquid.In another embodiment, described brazilwood extract dyeing liquid is made up of the water of 0.6% (w/v) haematine, 0.06% (w/v) sodium iodate, 5.28 (w/v) aluminium sulphate, 25% (v/v) ethanol, 6% (v/v) glacial acetic acid and surplus.
In one embodiment, described orange G dyeing liquor is OG-6.In a specific embodiment, described OG-6 dyeing liquor is made up of 0.3% (w/v) orange G, 0.015% (w/v) phosphoric acid tungsten hydrate, 4.05% (v/v) methyl alcohol, 80.95% (v/v) ethanol and deionized water.
In one embodiment, described EA dyeing liquor is made into by the dyestuff such as Yihong, BG, can be the EA dyeing liquor of EA36, EA50, EA65 or improvement.In one embodiment described EA-65 dyeing liquor, 0.1% (w/v) Yihong yellowish by 0.03% (w/v) Light Green SF, 0.2% (w/v) phosphoric acid tungsten, 2% (v/v) glacial acetic acid, 25% (v/v) pure methyl alcohol, 70% (v/v) 95% the pure water of ethanol and surplus form.
In one embodiment, EA dyeing liquor described herein is the EA dyeing liquor of improvement, and it is made up of the deionized water of 0.017% (w/v) Light Green SF, 0.05% (w/v) Bismarck, 0.225% (w/v) Yihong, 0.2% (w/v) phosphoric acid tungsten, 0.05% (v/v) lithium carbonate, 4.5% (v/v) methyl alcohol, 90.5% (v/v) ethanol and surplus.
In one embodiment, the dyeing of described intracellular acidic phosphatase be based on intracellular acid phosphatase under acid prerequisite, phosphoric acid naphthols AS-BI in matrix is hydrolyzed, release naphthols AS-BI, naphthols AS-BI and diazonium salt, form insoluble red precipitate, be positioned to realize in kytoplasm.In one embodiment, the pack for the dyeing of described acid phosphatase contains naphthols AS-BI phosphate base fluid and garnet diazo salt base fluid.In another embodiment, the reagent of the dyeing of described acid phosphatase also comprises sodium citrate buffer solution and sodium nitrite damping fluid.In one specific embodiment, described naphthols AS-BI phosphate base fluid is that 15mg/ml is dissolved in DMF.In a specific embodiment, described garnet diazo salt base fluid is that 10mg/ml fast red GBC (Fast Garnet GBC) is dissolved in 2-methyl cellosolve and hydrochloric acid.In one specific embodiment, the concentration of described acetate buffer is 2.0mol/L, pH=5.0.In one specific embodiment, described sodium nitrite damping fluid is that sodium nitrite 40mg/ml is dissolved in pure water and Tween20 (0.09% (v/v)).
In one embodiment, colouring method as herein described comprises dyeing cell after fixing being carried out in the staining reagent comprising naphthols AS-BI phosphate base fluid, garnet diazo salt base fluid, sodium citrate buffer solution and sodium nitrite damping fluid acid phosphatase.In another embodiment, colouring method as herein described comprises the cell of the dyeing through acid phosphatase is carried out pap staining.
In one embodiment, described pap staining comprises nuclear targeting and the endochylema dyeing of brazilwood extract dyeing, and endochylema dyeing is also called that in Pasteur's method OG-EA dyes, and it comprises orange G (OG) and the dyeing of EA two parts.In one embodiment, the immerged time of described brazilwood extract dyeing liquid is generally at 3-5min.In another embodiment, described brazilwood extract dyeing also comprises differentiation and alkalinization, can use hydrochloride alcohol differentiation and unsaturated carbonate lithium or the 0.5-3% liquid ammonia alkalinization of 0.25%, 0.5% or 1%, object point to melt haematoxylin that endochylema is stained with and returns indigo plant, and the time is about the several seconds.
In one embodiment, because orange G is a kind of small molecule dyes, can act on endochylema soon, the ordinary stain time is unsuitable long, is generally 1-3min.In one embodiment, the dyeing time of described EA dyeing is not higher than 3min.In particular embodiments, the dyeing time of the EA dyeing liquor of improvement disclosed herein be 10 seconds to 1min, or 20 to 50 seconds, such as 10,20,30,40,50 and 60 seconds.
In some embodiments, before enforcement tissue staining as herein described, described cervical squamous cells is fixed.In one embodiment, the immobile liquid for fixed cell is the immobile liquid in the pap staining of routine, such as 95% alcohol.In another embodiment, after the film-making of sample smear completes, take advantage of sample fresh and moistening time, put into the stationary cylinder filling 95% alcohol immediately, fixing 15-30min.Set time is no more than 1 week usually.
In one embodiment, the described immobile liquid for fixed cell is special immobile liquid disclosed herein, and it comprises A liquid and B liquid two parts, and A liquid is sodium citrate buffer solution, and B liquid is acetone, formaldehyde hybrid working liquid.In a specific embodiment, described special immobile liquid A liquid is the sodium citrate buffer solution of 0.05-0.2,0.8-0.15 or 0.1M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0.In another specific embodiment, described special immobile liquid B liquid contain 50%-70%, 50%, 55%, 60%, 65% or 70% acetone and 0.5-3%, 0.5,1.0,1.5,2.0 or 3.0% formalin (37-40% formalin).In another specific embodiment, described special immobile liquid A liquid is the sodium citrate buffer solution of 0.1M/L, and pH is 3.0; Described special immobile liquid B liquid contains 70% acetone and 1.0% formalin.
In another embodiment, colouring method described herein also comprises the step of fixing sample being carried out to conventional transparent and mounting.
In some embodiments, the sample using special immobile liquid disclosed herein fixing can preserve at least 1-2 week in 4 DEG C of refrigerators, preserves 1 week at normal temperatures, and the follow-up Color of not obvious impact and cellular morphology.
In another embodiment, use special immobile liquid disclosed herein that follow-up Color can be made more clear, the Color of acid phosphatase especially can be made more clear.
In another embodiment, the cell using special immobile liquid disclosed herein can make to fix not easily comes off from sample smear.
In some embodiments, method disclosed herein or kit, through clinical practice, compared with traditional Papanicolaou's vaginal smear technique, the sensitivity of diagnosis cervical squamous cells cancer or precancerous lesion improves.
In another embodiment, described sensitivity improves at least about 10%, 20% or 30%.
In some embodiments, method disclosed herein or kit, through clinical practice, compared with traditional Papanicolaou's vaginal smear technique, the false negative rate of diagnosis cervical squamous cells cancer or precancerous lesion reduces.
In another embodiment, described false positive rate is reduced to nearly 5% or 0%.
In other embodiments, utilize method disclosed herein or kit, the ANOMALOUS VARIATIONS that cervical squamous cells is very early stage can be found.
In embodiments, the pap staining liquid of improvement disclosed herein and colouring method, pap staining result is made to present blue background in whole cell dyeing, maintain the Color of pap staining to eucaryotic cell structure and level simultaneously, make the Color of acid phosphatase can clearly after improvement pap staining effect under show.
Utilize the result of this technology for detection to show CAP in abnormal cervical squamous cell kytoplasm and have activity expression, under its effect is presented at improved pap staining background, in abnormal squamous cell endochylema, CAP activity expression presents the sediment of red granules shape, and normal epithelium cell always shows feminine gender.This research specify that the activity expression of CAP comprised in cervical epithelial cells each stage paraplasm: ASC-US, ASC-H, LSIL, HSIL and SCC occur the positive.
It will be appreciated by those skilled in the art that, term disclosed herein " embodiment ", " embodiment " " another embodiment ", " some embodiments ", " other embodiments " and " specific embodiment ", only for illustrative purposes, illustrating working of an invention mode disclosed herein, is not the restriction to disclosed invention.When realizing in invention disclosed herein, those skilled in the art to its amendment and can replace.And when realizing invention disclosed herein, the element in above-mentioned embodiment, reagent, step and method can at random combine and replace.
Following examples are the exemplary illustrations to disclosed invention, can not understand the restriction to described invention.
Embodiment
Specimen origin:
All clinical samples all derive from First Attached Hospital, Anhui Medical Univ., by this institute professional and technical personnel according to gynaecology's routine operation collect specimen and smear.Hela cell is purchased from Shanghai Inst. of Life Science, CAS.
Materials and methods
1.
immobile liquid
special immobile liquid: comprise A, B liquid two parts.
A liquid: sodium citrate buffer solution 0.1M/L, pH=3.0
B liquid: acetone, formaldehyde hybrid working liquid, that is, 29% distilled water, 70% acetone (acetone) and 1% formalin (37-40% formaldehyde).
conventional immobile liquid: 95% alcohol or 95% alcohol and ether equivalent mixed liquor.
2.
zymochemistry dyeing liquor
Comprise A, B, C and D liquid
A liquid: acetate buffer: 2.0mol/L, pH=5.0
B liquid: naphthols AS-BI phosphate base fluid: 15mg/ml is dissolved in DMF
C liquid: garnet diazo salt base fluid: 10mg/ml fast red GBC (Fast Garnet GBC) is dissolved in 2-methyl cellosolve and hydrochloric acid
D liquid: sodium nitrite damping fluid: sodium nitrite 40mg/ml is dissolved in pure water and Tween20 (0.09% (v/v)).
3.
pap staining liquid
modified pap staining liquid:
Brazilwood extract dyeing liquid: the deionized water of 0.6% (w/v) haematine, 0.06% (w/v) sodium iodate, 5.28 (w/v) aluminium sulphate, 25% (v/v) ethanol, 6% (v/v) glacial acetic acid, surplus.
OG-6 dyeing liquor: the deionized water of 0.3% (w/v) orange G, 0.015% (w/v) phosphoric acid tungsten hydrate, 4.05% (v/v) methyl alcohol, 80.95% (v/v) ethanol and surplus.
Modified EA dyeing liquor: the deionized water of 0.017% (w/v) pale green, 0.05% (w/v) Bismarck, 0.225% (w/v) Yihong, 0.2% (w/v) phosphoric acid tungsten, 0.05% (v/v) lithium carbonate, 4.5% (v/v) methyl alcohol, 90.5% (v/v) ethanol, surplus.
contrast pap staining liquid:
Brazilwood extract dyeing liquid: the deionized water of 0.6% (w/v) haematine, 0.06% (w/v) sodium iodate, 5.28 (w/v) aluminium sulphate, 25% (v/v) ethanol, 6% (v/v) glacial acetic acid, surplus.
OG-6 dyeing liquor: the deionized water of 0.3% (w/v) orange G, 0.015% (w/v) phosphoric acid tungsten hydrate, 4.05% (v/v) methyl alcohol, 80.95% (v/v) ethanol and surplus.
Conventional EA-50 dyeing liquor: 3% BG aqueous solution 5ml, 20% Eosin Y 10ml, 0.2% (w/v) phosphoric acid tungsten 1g, glacial acetic acid 10ml, pure methyl alcohol 125ml, 95% ethanol 350ml.Lithium carbonate saturated solution is dripped after mixing.
Embodiment 1 is fixed
conventional fixing:
Smear (time not dry) is put into 95% alcohol or 95% alcohol and ether equivalent mixed liquor and fix at least 15 minutes.
Insert half a minute in 80%, 70%, 50% alcohol successively.Then insert with distilled water in more than 2 minutes.
special immobile liquid is fixed
Sample smear is fixed: get 7.5ml A liquid and add 42.5ml B liquid and be mixed into special immobile liquid.Rapidly sample smear is put into described immobile liquid 50 seconds, take out upper and lower rinsing 10 times in purifying water vat immediately.Can preserve 2 weeks in 4 DEG C of refrigerators.
Embodiment 2 dyes
One, traditional Papanicolaou staining procedure
(1) the sample smear 5-10 minute after fixing with haematoxylin dyeing, to karyon painted obviously, then clean with distilled water flushing.
(2) slough haematoxylin unnecessary in endochylema with 0.25% or 0.5% hydrochloride alcohol, transfer to light red to smear, at once clean with distilled water flushing, operation must rapidly, usual a few second.If the long meeting of bleaching time makes haematoxylin all disappear.
(3) put in unsaturated carbonate lithium solution and alkalize about 1 minute, transfer to smear light blue, then clean with distilled water.
(4) half a minute in 50%, 70%, 80% alcohol is inserted successively.Then at least 2 minutes are inserted in 95% alcohol with dehydration.
(5) in orange G-6 dye liquor, 3 minutes are contaminated.
(6) each upper and lower rinsing 6 times in two 95% alcohol cylinders respectively.
(7) in EA-50 dye liquor, 2-3 minute is contaminated to the painted distinctness of endochylema.
(8) remove unnecessary color two, three times with 95% ethanol wash, then put in absolute alcohol and anhydrate, then in dimethylbenzene, wash twice, finally use gummy sealing.
Two, new method dyeing: the staining procedure that enzyme dyeing is combined with improvement pap staining
acid phosphatase stain
(1) in staining jar, add the deionized water that 46ml has been heated to 37 DEG C, then add 2.5mlA liquid.In 1.5ml or 2ml centrifuge tube, add 10 B liquid and 10 C liquid and jog places at room temperature 3 minutes after 30 seconds, pour in the staining bottle containing A liquid and shake up.
(2) 10 D liquid are added in the staining jar of step (1) mix, fixed preparation smear is put into staining jar and covered cylinder cap and puts into 37 DEG C of constant water bath box lucifuges 45 minutes.
(3) sample smear is taken out, rinsing 5 minutes under tap water, then in pure water after rinsing once, to forward in PBS damping fluid rinsing to 10 times, then rinsing is once gently under tap water.
the pap staining of improvement
(1) the sample smear after acid phosphatase stain is put into the staining jar 5 minutes filling brazilwood extract dyeing liquid.
(2) take out sample slice, tap water rinse, then uses purified water rinsing under tap water after rinsing in 0.5% ammoniacal liquor (Fresh) cylinder upper and lower for 3 times, more each upper and lower rinsing 6 times in the alcohol cylinder of 50%, 70%, 80% and 95% respectively.
(3) sample slice is put into the staining jar 3 minutes containing OG-6 dyeing liquor, each upper and lower rinsing 6 times in two 95% alcohol cylinders respectively.
(4) sample slice is put into staining jar 15 second containing modified EA dyeing liquor, more each upper and lower rinsing 6 times in two 95% alcohol cylinders respectively.
(5) take out sample slice under tap water after rinsing in 0.5% ammoniacal liquor (Fresh) cylinder upper and lower for 3 times.
(6) staining jar put into after sample slice tap water rinse containing tap water is placed 1 hour.Take out mounting after dry and get final product read tablet.
for the diagnostic criteria of pathology doctor reference
1., owing to comprising gland cell, columnar cell and monocyte at cervical canal inner cell all containing highly active acid phosphatase expression of enzymes, therefore first when assessing NM-PAP coloration result need be distinguished cytomorphology and be ignored.
2. occur that red granules shape sediment is considered as the positive in pair ripe epithelial cell, middle layer cells and nearly basal layer cell endochylema, simultaneously according to the increase of its core and the abnormal TBS according to calendar year 2001 revision in addition classification by stages:
(1) Atypical squalors cell (ASC):
(I) Atypical squalors cell (ASC-US) of interrogatory,
(II) not except the Atypical squalors cell (ASC-H) of epithelium inner height pathology;
(2) low Squamous cell lesions (LSIL);
(3) high-grade squamous epithelial lesion (HSIL);
(4) squamous cell carcinoma (SCC).
3. suggestion is slight but have the NM-PAP positive expression case that positive cell number is less than 3 simultaneously to give the diagnostic result of " the early stage slight abnormality of epithelial cell " to cellular morphology change.
Result
Fig. 1, Fig. 2 are the Normal squamous epithelium of traditional pap staining display.Different colors is presented because of the acidophilia of squamous cell and basophilous difference.
Fig. 3, Fig. 4 are the Normal squamous epithelium of independent modified pap staining display.Visible unified blue background, eucaryotic cell structure and clear layer.
Fig. 5, Fig. 6 are the Atypical squalors cell (ASC-US) of the interrogatory of new method dyeing display.Therefrom have red granules shape sediment in high-visible abnormal cell endochylema, karyon compares with normal cell obvious increase.
Fig. 7 is new method dyeing display squamous cell abnormal in early stage, and whole smear only has this individual cells abnormal, and visible endochylema red granules precipitation and karyon increase.
Fig. 8 is that new method dyeing display dermoid cancer venereal disease becomes (SSC), and gather in endochylema red granules sediment, and karyon obviously increases.
The colour developing of the enzyme of Fig. 9 Hela cell after traditional immobile liquid (95% alcohol) is fixing, its colour developing is not good.
The enzyme colour developing of Figure 10 Hela cell after special immobile liquid is fixed, chemical staining presents red granules.
Above result shows and through clinical verification, acid phosphatase stain disclosed herein combines the pap staining of improvement, this new colouring method is compared with traditional Papanicolaou's vaginal smear technique, and the diagnosis susceptibility of cervical squamous cells cancer or precancerous lesion improves, and can be increased to about 30%.Or new colouring method disclosed herein can make the false negative rate of the diagnosis of cervical squamous cells cancer or precancerous lesion be reduced to nearly 0%.New immobile liquid disclosed herein can guarantee the above-mentioned effect of described new colouring method further, can shorten the set time, maybe can improve Color, such as, improve the Color of acid phosphatase.In addition, described new method can also find the ANOMALOUS VARIATIONS that cervical squamous cells is very early stage, and this simple change according to cytomorphology in the diagnosis of traditional Papanicolau staining process almost cannot realize.
Claims (28)
1. cervical squamous cells colouring method, comprises
The reagent utilizing cellular acid phosphatase to dye carries out acid phosphatase stain to described cervical squamous cells; And
Pap staining reagent is utilized to carry out pap staining to described cervical squamous cells, wherein said pap staining pack is containing the EA dyeing liquor of improvement, and it is made up of the deionized water of 0.017% (w/v) Light Green SF, 0.05% (w/v) Bismarck, 0.225% (w/v) Yihong, 0.2% (w/v) phosphoric acid tungsten, 0.05% (v/v) lithium carbonate, 4.5% (v/v) methyl alcohol, 90.5% (v/v) ethanol and surplus.
2. cervical squamous cells staining kit, comprises
The reagent of cellular acid phosphatase dyeing; And
Pap staining reagent, wherein said pap staining pack is containing the EA dyeing liquor of improvement, and it is made up of the deionized water of 0.017% (w/v) Light Green SF, 0.05% (w/v) Bismarck, 0.225% (w/v) Yihong, 0.2% (w/v) phosphoric acid tungsten, 0.05% (v/v) lithium carbonate, 4.5% (v/v) methyl alcohol, 90.5% (v/v) ethanol and surplus.
3. the diagnostic kit of individual cervical squamous cells cancer, described kit comprises,
The reagent of fixing individual cervical squamous cells sample;
The reagent of cellular acid phosphatase dyeing;
Pap staining reagent, wherein said pap staining pack is containing the EA dyeing liquor of improvement, it is made up of the deionized water of 0.017% (w/v) Light Green SF, 0.05% (w/v) Bismarck, 0.225% (w/v) Yihong, 0.2% (w/v) phosphoric acid tungsten, 0.05% (v/v) lithium carbonate, 4.5% (v/v) methyl alcohol, 90.5% (v/v) ethanol and surplus, and
Optionally comprise the instructions instructing pathology professional and technical personnel to judge staining cell.
4., for the fixating reagent of cervical squamous cells dyeing, described fixating reagent is made up of A liquid and B liquid, and wherein A liquid is sodium citrate buffer solution; Be acetone, formaldehyde hybrid working liquid with B liquid.
5. method as claimed in claim 1, wherein said cervical squamous cells comprises normal and abnormal cervical squamous cells.
6. method as claimed in claim 5, wherein said abnormal cervical squamous cells comprises the cervical squamous cells of canceration and the cervical squamous cells of precancerous lesion.
7. method described in claim 1 or 5, wherein said pap staining reagent also comprises brazilwood extract dyeing liquid, orange G (OG) dyeing liquor.
8. method as described in claim 1 or 5, the dyeing time of the EA dyeing liquor of wherein said improvement be 10 seconds to 1min.
9. method as described in claim 1 or 5, the dyeing time of the EA dyeing liquor of wherein said improvement is 20 to 50 seconds.
10. method as claimed in claim 8, wherein said dyeing time is 10,20,30,40,50 and 60 seconds.
11. as described in claim 1 or 5 method, wherein said method also comprises before the dyeing of described cervical squamous cells, is fixed described cervical squamous cells with fixating reagent.
12. methods as claimed in claim 11, wherein said fixating reagent is fixating reagent described in claim 4.
13. methods as claimed in claim 12, wherein
Described A liquid is the sodium citrate buffer solution of 0.05-0.2M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%-70% acetone and 0.5-3% formalin.
14. methods as claimed in claim 12, wherein
Described A liquid is the sodium citrate buffer solution of 0.8-0.15M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%-70% acetone and 0.5-3% formalin.
15. methods as claimed in claim 12, wherein
Described A liquid is the sodium citrate buffer solution of 0.1M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%, 55%, 60%, 65% or 70% acetone and 0.5%, 1.0%, 1.5%, 2.0% or 3.0% formalin.
16. kits as claimed in claim 2, wherein said cervical squamous cells comprises normal and abnormal cervical squamous cells.
17. kits as claimed in claim 16, wherein said abnormal cervical squamous cells comprises the cervical squamous cells of canceration and the cervical squamous cells of precancerous lesion.
18. as described in Claims 2 or 3 kit, wherein said pap staining reagent also comprises brazilwood extract dyeing liquid, orange G (OG) dyeing liquor.
19. as described in Claims 2 or 3 kit, the dyeing time of the EA dyeing liquor of wherein said improvement be 10 seconds to 1min.
20. as described in Claims 2 or 3 kit, the dyeing time of the EA dyeing liquor of wherein said improvement is 20 to 50 seconds.
21. kits as claimed in claim 19, wherein said dyeing time is 10,20,30,40,50 and 60 seconds.
22. as described in Claims 2 or 3 kit, wherein said kit also comprises fixating reagent.
23. kits as claimed in claim 22, wherein said fixating reagent is fixating reagent according to claim 4.
24. kits as claimed in claim 23, wherein
Described A liquid is the sodium citrate buffer solution of 0.05-0.2M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%-70% acetone and 0.5-3% formalin.
25. kits as claimed in claim 23, wherein
Described A liquid is the sodium citrate buffer solution of 0.8-0.15M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%-70% acetone and 0.5-3% formalin.
26. kits as claimed in claim 23, wherein
Described A liquid is the sodium citrate buffer solution of 0.1M/L, and pH is 2.0,2.5,3.0,3.5 or 4.0; And/or
Described B liquid contains 50%, 55%, 60%, 65% or 70% acetone and 0.5%, 1.0%, 1.5%, 2.0% or 3.0% formalin.
27. fixating reagents as claimed in claim 4, wherein said cervical squamous cells comprises normal and abnormal cervical squamous cells.
28. fixating reagents as claimed in claim 27, wherein said abnormal cervical squamous cells comprises the cervical squamous cells of canceration and the cervical squamous cells of precancerous lesion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110302187.7A CN103033409B (en) | 2011-10-09 | 2011-10-09 | The histocyte colouring method improved and application thereof |
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| CN103558150B (en) * | 2013-09-18 | 2015-12-23 | 石河子大学 | A kind of preparation method improveing Pap smear |
| 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 |
| CN103571909A (en) * | 2013-10-24 | 2014-02-12 | 温州市康泰生物科技有限公司 | Papanicolaou staining fluid and using method thereof |
| 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 |
| CN105842037B (en) * | 2016-03-21 | 2018-07-31 | 山东农业大学 | Colouring method that is a kind of while showing mast cell and acidophic cell |
| CN110926909A (en) * | 2019-12-23 | 2020-03-27 | 苏州堪赛尔生物技术有限公司 | Papanicolaou staining kit and staining method thereof |
| CN113607534B (en) * | 2021-08-20 | 2022-10-18 | 河南赛诺特生物技术有限公司 | Dyeing method, kit and application |
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