CN103033409A - Improved histiocyte staining method and application thereof - Google Patents
Improved histiocyte staining method and application thereof Download PDFInfo
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Abstract
The invention relates to an improved cervical squamous epithelial cell staining method, a cervical squamous epithelial cell staining kit and application of the method and the kit to diagnosis of individual cervical squamous epithelial cell cancers. The improved cervical squamous epithelial cell staining method comprises the steps of staining cervical squamous epithelial cells by using cell acid phosphatase staining reagent and staining the cervical squamous epithelial cells by using Pap staining reagent. The cervical squamous epithelial cell staining kit comprises the cell acid phosphatase staining reagent and the improved Pap staining reagent. The invention additionally relates to a kit for diagnosing individual cervical squamous epithelial cell cancers or precancerous lesions.
Description
Technical field
The application relates to histology and cytology colouring method and application thereof, 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 women's malignant tumour, and 500,000 cervical carcinoma new cases are whenever arranged every year, and wherein 80% case occurs in developing country.Every year new cases 13.15 ten thousand examples are arranged in China, account for 28.8% of world's cervical carcinoma new cases sum.In recent years, there is research to find the trend that the young case load of cervical carcinoma has to be 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 at present prevention and early diagnosis cervix cancer.
Since cell smear Papanicolaous vaginal smear technique (Pap-test) birth of the George Papanicolaou forties in 20th century, the 5 grades of classification of Pasteur that continue 40 years make the incidence of disease of cervix cancer reduce 70%-80%, use very universal because of its cost-effectiveness height in developing country simultaneously.But owing to be subjected to many factors, false positive rate and the false negative rate of Papanicolaous vaginal smear technique are too high.Invented in recent years a lot of new methods and new technology, having comprised: Thinprep technology (Thin Prep, Liquid-BasedMonolayers TCT)-be intended to the improve technology of slice-making quality; Fluorescence is inspected method (Speculoscopy)-luminous amplifying technique of applied chemistry in conjunction with the method for 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, Papanicolaous vaginal smear technique is still the Main Means of present examination cervical carcinoma.
Summary of the invention
First aspect, this paper relates to improved cervical squamous cells colouring method, comprises
Described cervical squamous cells is carried out acid phosphatase stain; And
Described cervical squamous cells is carried out pap staining.
Second aspect, this paper relates to improved cervical squamous cells staining kit, comprises
The reagent of cell acid phosphatase stain; And
Pap staining reagent.
The third aspect, this paper relates to the method for diagnosing individual cervical squamous cells cancer, and described method comprises,
Collect individual cervical squamous cells sample,
Fixing described sample,
Described cervical squamous cells is carried out acid phosphatase stain,
Described cervical squamous cells is carried out pap staining, and
Judge according to the colour developing result whether cell is cancer cell or precancerous lesion by the pathology professional and technical personnel.
Fourth aspect, this paper relates to the diagnostic kit of individual cervical squamous cells cancer, and described kit comprises,
The reagent of the cervical squamous cells sample of fixing individuality;
The reagent of cell acid phosphatase stain;
Pap staining reagent, and
Randomly comprise the instructions that instructs the pathology professional and technical personnel that staining cell is judged.
The 5th aspect, this paper relates to special immobile liquid, and described immobile liquid comprises
A liquid: sodium citrate buffer solution; With
B liquid: acetone, formaldehyde hybrid working liquid.
Description of drawings
Fig. 1, Fig. 2 are the normal squamous cell (Fig. 1, Fig. 2 are respectively 20X and 40X) that traditional pap staining shows.Acidophilia and the basophilous different different colors that present because of squamous cell.
Fig. 3, Fig. 4 are the normal squamous cell (Fig. 3, Fig. 4 are respectively 10X and 20X) that the modified pap staining shows.
Fig. 5, Fig. 6 are that acid phosphatase stain is in conjunction with the squamous cell that is not true to type (ASC-US) of the interrogatory of the new method dyeing demonstration of improved pap staining.
Fig. 7 is that acid phosphatase stain is in conjunction with the new method dyeing demonstration squamous cell abnormal in early stage of improved pap staining.
Fig. 8 is that acid phosphatase stain is in conjunction with the new method dyeing demonstration dermoid cancer venereal disease change (SCC) of improved pap staining.
Fig. 9 shows the enzyme dyeing of Hela cell after traditional immobile liquid is fixing.
Figure 10 shows the enzyme dyeing of Hela cell after special immobile liquid is fixing.
Embodiment describes in detail
The inventor is surprised to find, and it is active simultaneously in conjunction with improved pap staining technology to utilize Cytochemical staining method to detect cervical cell acid phosphatase (CAP), can improve the unusual Color of cervical squamous cells.
Accordingly, first aspect, this paper relates to improved cervical squamous cells colouring method, comprises
Described cervical squamous cells is carried out acid phosphatase stain; And
Described cervical squamous cells is carried out pap staining.
Second aspect, this paper relates to improved cervical squamous cells staining kit, comprises
The reagent of cell acid phosphatase stain; And
Pap staining reagent.
The third aspect, this paper relates to the method for diagnosing individual cervical squamous cells cancer, and described method comprises,
Collect individual cervical squamous cells sample;
Fixing described sample;
Described cervical squamous cells is carried out acid phosphatase stain;
Described cervical squamous cells is carried out pap staining; And
Judge according to the colour developing result whether cell is cancer cell or precancerous lesion by the pathology professional and technical personnel.
Fourth aspect, this paper relates to the diagnostic kit of individual cervical squamous cells cancer, and described kit comprises,
The reagent of fixed sample;
The reagent of cell acid phosphatase stain;
Pap staining reagent, and
Randomly comprise the instructions that instructs the pathology professional and technical personnel that staining cell is judged.
The 5th aspect, this paper relates to special immobile liquid, 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 normal, unusual, canceration and precancerous lesion thereof.In another embodiment, described unusual, canceration or precancerous lesion cervical squamous cells comprise, squamous cell (ASC) is not true to type, it comprises the squamous cell that is not true to type (ASC-US) of (i) interrogatory, (ii) not except the squamous cell that is not true to type (ASC-H) of epithelium inner height pathology; Low scaly epithelium pathology (LSIL); Height scaly epithelium pathology (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-references, especially refer to female mammal or female patient.
In one embodiment, the pap staining reagent that is used for described method and kit comprises three kinds of dyeing liquors, i.e. brazilwood extract dyeing liquid, orange G (OG) dyeing liquor and EA (Eosin Azure) dyeing liquor.
In one embodiment, described brazilwood extract dyeing liquid can be Harris brazilwood extract dyeing liquid or Gill improvement brazilwood extract dyeing liquid.In another embodiment, described brazilwood extract dyeing liquid is comprised 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 comprised 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 dyestuffs such as Yihong, BGs, can be the EA dyeing liquor of EA36, EA50, EA65 or improvement.The pure water of 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, the pure methyl alcohol of 25% (v/v), 70% (v/v), 95% ethanol and surplus forms in one embodiment.
In one embodiment, EA dyeing liquor described herein is the EA dyeing liquor of improvement, and its deionized water by 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 forms.
In one embodiment, the dyeing of described cell Acid Phosphatase is based on intracellular acid phosphatase under acid prerequisite, phosphoric acid naphthols AS-BI in the matrix is hydrolyzed, discharge naphthols AS-BI, naphthols AS-BI and diazo salt coupling, form insoluble red precipitate, be positioned in the kytoplasm and realize.In one embodiment, the reagent that is used for the dyeing of described acid phosphatase comprises 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 the 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 the 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 that cell carries out the dyeing of acid phosphatase in the staining reagent that comprises naphthols AS-BI phosphate base fluid, garnet diazo salt base fluid, sodium citrate buffer solution and sodium nitrite damping fluid with fixing afterwards.In another embodiment, colouring method as herein described comprises the cell through the dyeing of acid phosphatase is carried out pap staining.
In one embodiment, described pap staining comprises nucleus dyeing and the endochylema dyeing of brazilwood extract dyeing, and endochylema dyeing also is called OG-EA dyeing in Pasteur's method, 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 alkalization process, can use the differentiation of 0.25%, 0.5% or 1% hydrochloride alcohol and unsaturated carbonate lithium or 0.5-3% liquid ammonia alkalinization, purpose is minute to melt the haematoxylin that is stained with on the endochylema and return indigo plant, pact several seconds time.
In one embodiment, because orange G is a kind of little molecular dye, can act on soon endochylema, the ordinary stain time is unsuitable long, is generally 1-3min.In one embodiment, the dyeing time of described EA dyeing is not for being higher than 3min.In specific embodiment, the dyeing time of the EA dyeing liquor of improvement disclosed herein be 10 seconds to 1min, or 20 to 50 seconds, for example 10,20,30,40,50 and 60 seconds.
In some embodiments, before implementing tissue staining as herein described, described cervical squamous cells is fixed.In one embodiment, the immobile liquid that is used for fixed cell is the immobile liquid of the pap staining of routine, for example 95% alcohol.In another embodiment, after the film-making of sample smear is finished, take advantage of sample when fresh and moistening, put into immediately the stationary cylinder that fills 95% alcohol, fixedly 15-30min.Set time was no more than for 1 week usually.
In one embodiment, described immobile liquid for fixed cell is special immobile liquid disclosed herein, and it comprises A liquid and two parts of B liquid, 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 contains 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 conventional transparent and mounting.
In some embodiments, use the fixing sample of special immobile liquid disclosed herein can in 4 ℃ of refrigerators, preserve at least 1-2 week, preserved at normal temperatures for 1 week, and follow-up Color and the cellular morphology of not obvious impact.
In another embodiment, use the special immobile liquid disclosed herein can be so that follow-up Color is more clear, especially can be so that the Color of acid phosphatase be more clear.
In another embodiment, use special immobile liquid disclosed herein can make fixing cell be difficult for coming off from the sample smear.
In some embodiments, method disclosed herein or kit through clinical practice, are compared with traditional Papanicolaous vaginal smear technique, and 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, are compared with traditional Papanicolaous vaginal smear technique, and 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, can find the ANOMALOUS VARIATIONS that cervical squamous cells is very early stage.
In embodiments, pap staining liquid and the colouring method of improvement disclosed herein, so that the pap staining result presents blue background in whole cell dyeing, the Color of acid phosphatase kept simultaneously the Color of pap staining to eucaryotic cell structure and level, so that can show clearly under the effect of pap staining after the improvement.
Utilize the result of this technology for detection to show that CAP has activity expression in the unusual cervical squamous cells kytoplasm, its effect is presented under the improved pap staining background, the CAP activity expression presents the sediment of red granules shape in the unusual squamous cell endochylema, and normal epithelium cell always shows feminine gender.The activity expression of the clear and definite CAP of this research comprises at the paraplasm stages of cervical epithelial cells: the positive appears in ASC-US, ASC-H, LSIL, HSIL and SCC.
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 be for illustrative purposes, having exemplified working of an invention mode disclosed herein, is not the restriction to disclosed invention.In the situation that can be implemented in invention disclosed herein, those skilled in the art can be to its modification and replacement.And in the situation that can realize invention disclosed herein, the element in the above-mentioned embodiment, reagent, step and method can at random make up 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 the professional and technical personnel of this institute according to gynaecology's routine operation collect specimen and smear.The Hela cell is available from Shanghai Inst. of Life Science, CAS.
Materials and methods
1.
Immobile liquid
Special immobile liquid: comprise A, two parts of B liquid.
A liquid: sodium citrate buffer solution 0.1M/L, pH=3.0
B liquid: acetone, formaldehyde hybrid working liquid, that is, and 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.
The 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 the DMF
C liquid: garnet diazo salt base fluid: 10mg/ml fast red GBC (Fast Garnet GBC) is dissolved in 2-methyl cellosolve and the 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.Drip the lithium carbonate saturated solution after mixing.
Embodiment 1 is fixing
Conventional fixing:
Smear when not dried () was put into 95% alcohol or 95% alcohol and ether equivalent mixed liquor fixing at least 15 minutes.
Insert successively half a minute in 80%, 70%, 50% alcohol.Then insert with distilled water in more than 2 minutes.
Special immobile liquid is fixed
The sample smear is fixed: get 7.5ml A liquid and add 42.5ml B liquid and be mixed into special immobile liquid.Rapidly the sample smear is put into described immobile liquid 50 seconds, take out immediately in the purifying water vat up and down rinsing 10 times.Can in 4 ℃ of refrigerators, preserve for 2 weeks.
Embodiment 2 dyeing
One, traditional pap staining step
(1) the sample smear after fixing with haematoxylin dyeing 5-10 minute, to karyon painted obviously till, then clean with distilled water flushing.
(2) slough unnecessary haematoxylin in the endochylema with 0.25% or 0.5% hydrochloride alcohol, transfer to light redly, at once clean with distilled water flushing to smear, operation must be rapidly, common several seconds.If the long meeting of bleaching time all disappears haematoxylin.
(3) put in the unsaturated carbonate lithium solution about 1 minute of alkalization, transfer to light bluely to smear, clean with distilled water again.
(4) insert successively half a minute in 50%, 70%, 80% alcohol.Then insert in 95% alcohol at least 2 minutes with dehydration.
(5) in orange G-6 dye liquor, dyed 3 minutes.
(6) respectively each up and down rinsing 6 times in two 95% alcohol cylinders.
(7) in the EA-50 dye liquor, dye 2-3 minute till the painted distinctness of endochylema.
(8) remove unnecessary color two, three times with the washing of 95% alcohol, put in the absolute alcohol again and anhydrate, then in dimethylbenzene, wash twice, use at last gummy sealing.
Two, new method dyeing: the staining procedure that enzyme dyeing is combined with the improvement pap staining
Acid phosphatase stain
(1) in staining jar, adds 46ml and be heated to 37 ℃ deionized water, then add 2.5mlA liquid.In 1.5ml or 2ml centrifuge tube, add 10 B liquid and 10 C liquid and jog and placed at room temperature 3 minutes afterwards in 30 seconds, pour in the staining bottle that contains A liquid and shake up.
(2) with mixing in the staining jar of 10 D liquid adding steps (1), the fixed preparation smear is put into staining jar and is covered cylinder cap and put into 37 ℃ of constant water bath box lucifuges 45 minutes.
(3) the sample smear is taken out, rinsing is 5 minutes under tap water, again in pure water after the rinsing once, forwards in the PBS damping fluid rinsing to 10 times, and rinsing is once gently under tap water again.
The pap staining of improvement
(1) the sample smear behind the acid phosphatase stain is put into the staining jar 5 minutes that fills brazilwood extract dyeing liquid.
(2) take out sample slice, the purified water rinsing then use in the tap water rinsing under tap water after the rinsing in 0.5% ammoniacal liquor (fresh preparation) cylinder for 3 times up and down, more respectively 50%, 70%, in 80% and 95% the alcohol cylinder respectively about rinsing 6 times.
(3) sample slice is put into the staining jar 3 minutes that contains the OG-6 dyeing liquor, respectively each up and down rinsing 6 times in two 95% alcohol cylinders.
(4) sample slice is put into 15 seconds of staining jar that contain modified EA dyeing liquor, more respectively each up and down rinsing 6 times in two 95% alcohol cylinders.
(5) take out sample slice under tap water after the rinsing in 0.5% ammoniacal liquor (fresh preparation) cylinder for 3 times up and down.
(6) sample slice being put into the staining jar that contains tap water after with the tap water rinsing placed 1 hour.Taking out wait doing rear mounting is readable.
Diagnostic criteria for pathology doctor reference
1. owing to comprise that at the cervical canal inner cell gland cell, columnar cell and monocyte all contain highly active acid phosphatase expression of enzymes, therefore when assessment NM-PAP coloration result, at first need on cytomorphology, to be distinguished and ignore.
2. be considered as the positive to red granules shape sediment occurring in ripe epithelial cell, middle layer cells and the nearly basal layer cell endochylema, simultaneously according to the increase of its nuclear with unusually according to by stages in addition classification of the TBS of calendar year 2001 revision:
(1) squamous cell (ASC) that is not true to type:
(I) squamous cell that is not true to type (ASC-US) of interrogatory,
(II) not except the squamous cell that is not true to type (ASC-H) of epithelium inner height pathology;
(2) low scaly epithelium pathology (LSIL);
(3) height scaly epithelium pathology (HSIL);
(4) squamous cell carcinoma (SCC).
3. suggestion changes the diagnostic result that case slight but that have NM-PAP positive expression while positive cell number to be less than 3 gives " the early stage slight abnormality of epithelial cell " to cellular morphology.
The result
Fig. 1, Fig. 2 are the normal squamous cell that traditional pap staining shows.Acidophilia and the basophilous different different colors that present because of squamous cell.
Fig. 3, Fig. 4 are the normal squamous cell that independent modified pap staining shows.As seen unified blue background, eucaryotic cell structure and clear layer.
Fig. 5, Fig. 6 are the squamous cell that is not true to type (ASC-US) of the interrogatory of new method dyeing demonstration.Therefrom in the high-visible abnormal cell endochylema red granules shape sediment is arranged, karyon and normal cell relatively have obvious increase.
Fig. 7 is that new method dyeing shows the squamous cell abnormal in early stage, and whole smear only has this individual cells unusual, and visible endochylema red granules precipitation and karyon increase.
Fig. 8 is that new method dyeing shows that the dermoid cancer venereal disease becomes (SSC), the red granules sediment that gathers in the endochylema, 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 fixing, chemical staining presents red granules.
Above result shows and through clinical verification, acid phosphatase stain disclosed herein is in conjunction with the pap staining of improvement, this new colouring method is compared with traditional Papanicolaous vaginal smear technique, and the diagnosis susceptibility of cervical squamous cells cancer or precancerous lesion improves, and can be increased to about 30%.Perhaps, 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 further be guaranteed the above-mentioned effect of described new colouring method, can shorten the set time, maybe can improve Color, for example improves the Color of acid phosphatase.In addition, described new method can also be found the ANOMALOUS VARIATIONS that cervical squamous cells is very early stage, and this simple variation according to cytomorphology in the diagnosis of traditional Papanicolau staining process almost can't realize.
Claims (10)
1. the cervical squamous cells colouring method comprises
Utilize the reagent of cell acid phosphatase stain that described cervical squamous cells is carried out acid phosphatase stain; And
Utilize pap staining reagent that described cervical squamous cells is carried out pap staining.
2. the cervical squamous cells staining kit comprises
The reagent of cell acid phosphatase stain; And
Pap staining reagent.
3. the diagnostic kit of individual cervical squamous cells cancer, described kit comprise,
The reagent of fixing individual cervical squamous cells sample;
The reagent of cell acid phosphatase stain;
Pap staining reagent, and
Randomly comprise the instructions that instructs the pathology professional and technical personnel that staining cell is judged.
4. be used for the fixating reagent of cervical squamous cells dyeing, described fixating reagent comprises
A liquid: sodium citrate buffer solution; With
B liquid: acetone, formaldehyde hybrid working liquid.
5. as claimed in claim 1 method, the described kit of claim 2, the described immobile liquid of claim 4, wherein said cervical squamous cells comprises cervical squamous cells normal, unusual, canceration and precancerous lesion thereof.
6. each described method of claim 1-5, kit or immobile liquid, wherein said pap staining reagent comprises brazilwood extract dyeing liquid, orange G (OG) dyeing liquor and EA (Eosin Azure) dyeing liquor, wherein said EA dyeing liquor is the EA dyeing liquor of improvement, and it is by 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, form with the deionized water of surplus.
7. such as claim 1-6 method, kit or immobile liquid as described in each, the dyeing time of the EA dyeing liquor of wherein said improvement be 10 seconds to 1min, or 20 to 50 seconds, for example 10,20,30,40,50 and 60 seconds.
8. such as claim 1-5 method or kit as described in each, wherein said method comprises also that to before the described cervical squamous cells dyeing with fixating reagent described cervical squamous cells is fixed, perhaps, described kit also comprises fixating reagent.
9. as claimed in claim 8 method or kit, wherein said fixating reagent is the described fixating reagent of claim 4.
10. as claimed in claim 9 methods or kit, wherein
Described 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; And/or
Described B liquid contains 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.
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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 |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| 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 |
| CN112781963A (en) * | 2020-12-30 | 2021-05-11 | 深路医学科技(武汉)有限公司 | Papanicolaou staining solution and preparation method and staining method thereof |
| CN113607534A (en) * | 2021-08-20 | 2021-11-05 | 河南赛诺特生物技术有限公司 | Dyeing method, kit and application |
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| CN103558150A (en) * | 2013-09-18 | 2014-02-05 | 石河子大学 | Cervical carcinoma screening method |
| 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 |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| 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 |
| CN112781963A (en) * | 2020-12-30 | 2021-05-11 | 深路医学科技(武汉)有限公司 | Papanicolaou staining solution and preparation method and staining method thereof |
| CN112781963B (en) * | 2020-12-30 | 2024-04-30 | 深路医学科技(武汉)有限公司 | Papanicolaou staining solution and preparation method and staining method thereof |
| CN113607534A (en) * | 2021-08-20 | 2021-11-05 | 河南赛诺特生物技术有限公司 | Dyeing method, kit and application |
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