CN110926909A - Papanicolaou staining kit and staining method thereof - Google Patents
Papanicolaou staining kit and staining method thereof Download PDFInfo
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- CN110926909A CN110926909A CN201911334765.8A CN201911334765A CN110926909A CN 110926909 A CN110926909 A CN 110926909A CN 201911334765 A CN201911334765 A CN 201911334765A CN 110926909 A CN110926909 A CN 110926909A
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- 238000010186 staining Methods 0.000 title claims abstract description 34
- 238000007447 staining method Methods 0.000 title abstract description 9
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims abstract description 42
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000012192 staining solution Substances 0.000 claims abstract description 33
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 68
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 59
- 235000019441 ethanol Nutrition 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000012153 distilled water Substances 0.000 claims description 12
- 235000015110 jellies Nutrition 0.000 claims description 12
- 239000008274 jelly Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- HSXUHWZMNJHFRV-UHFFFAOYSA-L disodium;6-oxido-5-phenyldiazenyl-4-sulfonaphthalene-2-sulfonate Chemical compound [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1N=NC1=CC=CC=C1 HSXUHWZMNJHFRV-UHFFFAOYSA-L 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- 229940103272 aluminum potassium sulfate Drugs 0.000 claims description 9
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 6
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical class [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 3
- 230000018044 dehydration Effects 0.000 claims description 3
- 238000006297 dehydration reaction Methods 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 229910052808 lithium carbonate Inorganic materials 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 235000019991 rice wine Nutrition 0.000 claims description 3
- 239000012047 saturated solution Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 229940032753 sodium iodate Drugs 0.000 claims description 3
- 235000015281 sodium iodate Nutrition 0.000 claims description 3
- 239000011697 sodium iodate Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000008096 xylene Substances 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 31
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002344 surface layer Substances 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 210000004766 cell nucleus structure Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a Papanicolaou staining kit and a staining method thereof. The papanicolaou staining kit comprises a hematoxylin staining solution, an eosin staining solution and an orange G staining solution, wherein the pH value of the eosin staining solution is controlled to be 6.5-6.8, so that the eosin staining solution is stained in a weak acid environment, and the tinting strength of the eosin staining solution is improved. By adopting the scheme provided by the invention, the time from fixing to staining and mounting of the cell smear can be shortened, the detection efficiency is improved, and the detection efficiency is further improved and the detection difficulty is reduced due to the clear cell structure after staining.
Description
Technical Field
The invention relates to the field of in vitro diagnosis, in particular to a Papanicolaou staining kit and a staining method thereof.
Background
The Papanicolaou staining method is the most common staining method in pathological section and cast-off cell staining, and the staining method not only has the characteristics of clear cell nucleus structure, obvious color separation, good transparency, bright cytoplasm color and the like, but also can be used for preserving the stained specimen for a long time without decoloring easily.
The traditional Papanicolaou staining kit has complex components, complicated staining procedures, long time and difficult mastering. The technicians in the inspection generally feel the difficulty of operation.
Disclosure of Invention
In order to overcome the defects in the prior art, the embodiment of the invention provides a papanicolaou staining kit and a staining method thereof, which are used for solving at least one of the problems.
The embodiment of the application discloses: a Papanicolaou staining kit comprises hematoxylin staining solution, eosin staining solution and orange G staining solution, wherein,
the preparation method of the hematoxylin staining solution comprises the following steps: adding 2g of hematoxylin into 250ml of 95% ethanol to obtain hematoxylin alcohol solution; adding 17g of aluminum potassium sulfate into 750ml of distilled water, and stirring to completely dissolve the aluminum potassium sulfate to obtain an aluminum potassium sulfate solution; adding the hematoxylin alcohol solution into the aluminum sulfate solution, and adding 0.2g of sodium iodate into the solution; then adding 20ml of glacial acetic acid with the volume concentration of 95.5% into the solution;
the preparation method of the eosin dye solution comprises the following steps:
dissolving 2.5g of light green in 5ml of distilled water to obtain a light green solution, and adding the light green solution into 500ml of absolute ethyl alcohol to obtain light green alcohol solution;
adding 0.5g of Bowmember's jelly into 5ml of distilled water to obtain a Bowmember's jelly solution, and adding the Bowmember's jelly into 100ml of absolute ethanol to obtain Bowmember's jelly liquor;
adding 2.5g of eosin into 500ml of 95% ethanol to obtain eosin solution;
mixing 45ml of the light green alcohol extract, 10ml of the kojiki rice wine extract and 45ml of the eosin alcohol solution, and adding 0.2g of phosphotungstic acid into the mixed solution to obtain an eosin dye solution;
the preparation method of the orange G dye solution comprises the following steps:
0.5G of orange G is dissolved in 5ml of distilled water to obtain an orange solution, and 100ml of absolute ethyl alcohol is added into the orange solution to obtain the orange G dye solution.
Specifically, the papanicolaou staining kit comprises 20ml of hematoxylin staining solution, 20ml of eosin staining solution and 20ml of orange G staining solution.
Specifically, the preparation method of the eosin dye solution further comprises the following steps: several drops of saturated lithium carbonate solution were added to the eosin dye solution.
Specifically, the pH value of the eosin dye solution after the lithium carbonate saturated solution is added is 6.5-6.8.
Specifically, the preparation method of the orange G dye solution further comprises the following steps: 0.015G phosphotungstic acid was added to the orange G dye liquor.
The embodiment of the application also discloses: a method of staining with the papanicolaou staining kit of the present embodiment, comprising the steps of:
a. immersing the fixed cell smear into alcohol for 3min, and then washing with water;
b. b, immersing the cell smear obtained in the step a into hematoxylin staining solution for 2min, and then flushing for 5min by adopting running water;
c. b, immersing the cell smear obtained in the step b in 75%, 85% and 95% alcohol for 40s in sequence to dehydrate;
d. c, immersing the cell smear obtained in the step c into orange G staining solution for 1-2 min;
e. d, soaking and washing the cell smear obtained in the step d in 95% alcohol for 2 times;
f. e, staining the cell smear obtained in the step e for 4-8 min by using eosin staining solution;
g. and f, soaking and washing the cell smear obtained in the step f in 95% alcohol for 2 times, then placing the cell smear in absolute ethyl alcohol for dehydration, then placing the cell smear in xylene for washing and clearing, and then sealing the cell smear after clearing by using neutral gum.
Specifically, the step a comprises the following steps: the fixed cell smear was immersed in 80%, 70%, 50% alcohol for 1min each in turn, and then washed with water.
The invention has at least the following beneficial effects:
1. the preparation method of each component in the pasteur staining kit is simple and low in cost.
2. The Papanicolaou staining kit can shorten the time from fixing to staining and then to mounting of a cell smear, and improve the staining efficiency.
3. The pH value of the eosin dye solution is controlled to be 6.5-6.8, and the tinting strength of the eosin dye solution can be improved in the weak acid environment.
4. The Papanicolaou staining kit can enable a cell smear to display various colors, improves the definition of a stained cell structure, and is beneficial to detection of detection personnel.
In order to make the aforementioned and other objects, features and advantages of the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The papanicolaou staining kit in the embodiment comprises 20ml of hematoxylin staining solution, 20ml of eosin staining solution and 20ml of orange G staining solution, and the papanicolaou staining kit needs to be maintained in a normal-temperature and dark environment. The preparation method of the hematoxylin staining solution comprises the following steps:
adding 2g of hematoxylin into 250ml of 95% ethanol to obtain hematoxylin alcohol solution; adding 17g of aluminum potassium sulfate into 750ml of distilled water, and stirring to completely dissolve the aluminum potassium sulfate to obtain an aluminum potassium sulfate solution; adding the hematoxylin alcohol solution into the aluminum sulfate solution, and adding 0.2g of sodium iodate into the mixed solution; then, 20ml of glacial acetic acid with a volume concentration of 95.5% was added to the above solution.
The preparation method of the eosin dye solution comprises the following steps:
dissolving 2.5g of light green in 5ml of distilled water to obtain a light green solution, and adding the light green solution into 500ml of absolute ethyl alcohol to obtain light green alcohol solution;
adding 0.5g of Bowmember's jelly into 5ml of distilled water to obtain a Bowmember's jelly solution, and adding the Bowmember's jelly into 100ml of absolute ethanol to obtain Bowmember's jelly liquor;
adding 2.5g of eosin into 500ml of 95% ethanol to obtain eosin solution;
and mixing 45ml of the light green alcohol sperm, 10ml of the kojiki rice wine sperm and 45ml of the eosin alcohol solution, and adding 0.2g of phosphotungstic acid into the mixed solution to obtain an eosin dye solution.
Furthermore, in order to enable the pH value of the eosin dye solution to reach 6.5-6.8, a plurality of drops of lithium carbonate saturated solution can be added into the eosin dye solution. The weak acid environment can make the coloring power of eosin dye solution higher, and further improve the definition of the cell structure after dyeing.
The preparation method of the orange G dye solution comprises the following steps:
0.5G of orange G is dissolved in 5ml of distilled water to obtain an orange solution, and 100ml of absolute ethyl alcohol is added into the orange solution to obtain the orange G dye solution.
Furthermore, 0.015G of phosphotungstic acid can be added into the orange G dye solution to improve the tinting strength of the orange G dye solution.
The staining method of the cell smear by the Papanicolaou staining kit in the embodiment comprises the following steps:
a. the fixed cell smear was immersed in 80%, 70%, 50% alcohol for 1min each in turn, and then washed with water.
b. And (b) immersing the cell smear obtained in the step a into hematoxylin staining solution for 2min, and then washing for 5min by using running water.
c. The cell smear obtained in step b was immersed in 75%, 85%, 95% alcohol for 40s each in order to be dehydrated.
d. And d, immersing the cell smear obtained in the step c into orange G staining solution for 1-2 min.
e. The cell smear obtained in step d was rinsed 2 times in 95% alcohol.
f. And e, staining the cell smear obtained in the step e for 4-8 min by using eosin staining solution.
g. And f, soaking and washing the cell smear obtained in the step f in 95% alcohol for 2 times, then placing the cell smear in absolute ethyl alcohol for dehydration, then placing the cell smear in xylene for washing and clearing, and then sealing the cell smear after clearing by using neutral gum.
In the final cell smear obtained by the method, the cell nucleus is blue, the nucleolus is red, the cytoplasm of the bottom layer and the surface layer reticular nuclear cells are both dyed into blue-green, the cytoplasm of the surface layer condensed nuclear cells are dyed into light red or light blue-green, and the red cells are bright red or orange red.
The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (7)
1. A Papanicolaou staining kit is characterized by comprising hematoxylin staining solution, eosin staining solution and orange G staining solution, wherein,
the preparation method of the hematoxylin staining solution comprises the following steps: adding 2g of hematoxylin into 250ml of 95% ethanol to obtain hematoxylin alcohol solution; adding 17g of aluminum potassium sulfate into 750ml of distilled water, and stirring to completely dissolve the aluminum potassium sulfate to obtain an aluminum potassium sulfate solution; adding the hematoxylin alcohol solution into the aluminum sulfate solution, and adding 0.2g of sodium iodate into the solution; then adding 20ml of glacial acetic acid with the volume concentration of 95.5% into the solution;
the preparation method of the eosin dye solution comprises the following steps:
dissolving 2.5g of light green in 5ml of distilled water to obtain a light green solution, and adding the light green solution into 500ml of absolute ethyl alcohol to obtain light green alcohol solution;
adding 0.5g of Bowmember's jelly into 5ml of distilled water to obtain a Bowmember's jelly solution, and adding the Bowmember's jelly into 100ml of absolute ethanol to obtain Bowmember's jelly liquor;
adding 2.5g of eosin into 500ml of 95% ethanol to obtain eosin solution;
mixing 45ml of the light green alcohol extract, 10ml of the kojiki rice wine extract and 45ml of the eosin alcohol solution, and adding 0.2g of phosphotungstic acid into the mixed solution to obtain an eosin dye solution;
the preparation method of the orange G dye solution comprises the following steps:
0.5G of orange G is dissolved in 5ml of distilled water to obtain an orange solution, and 100ml of absolute ethyl alcohol is added into the orange solution to obtain the orange G dye solution.
2. The pap staining kit of claim 1 wherein 20ml of the hematoxylin stain, 20ml of the eosin stain, and 20ml of the orange G stain are included in the pap staining kit.
3. The papanicolaou staining kit according to claim 1, wherein the method for preparing the eosin staining solution further comprises: several drops of saturated lithium carbonate solution were added to the eosin dye solution.
4. The pasteurized staining kit of claim 3, wherein the pH of the eosin staining solution after the lithium carbonate saturated solution is added is between 6.5 and 6.8.
5. The papanicolaou staining kit according to claim 1, wherein the method for preparing the orange G staining solution further comprises: 0.015G phosphotungstic acid was added to the orange G dye liquor.
6. A method of staining using the Papanicolaou staining kit of any one of claims 1-5, comprising the steps of:
a. immersing the fixed cell smear into alcohol for 3min, and then washing with water;
b. b, immersing the cell smear obtained in the step a into hematoxylin staining solution for 2min, and then flushing for 5min by adopting running water;
c. b, immersing the cell smear obtained in the step b in 75%, 85% and 95% alcohol for 40s in sequence to dehydrate;
d. c, immersing the cell smear obtained in the step c into orange G staining solution for 1-2 min;
e. d, soaking and washing the cell smear obtained in the step d in 95% alcohol for 2 times;
f. e, staining the cell smear obtained in the step e for 4-8 min by using eosin staining solution;
g. and f, soaking and washing the cell smear obtained in the step f in 95% alcohol for 2 times, then placing the cell smear in absolute ethyl alcohol for dehydration, then placing the cell smear in xylene for washing and clearing, and then sealing the cell smear after clearing by using neutral gum.
7. The method of claim 6, wherein step a comprises: the fixed cell smear was immersed in 80%, 70%, 50% alcohol for 1min each in turn, and then washed with water.
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Address after: Unit 306, B1 Floor, 218 Xinghu Street, Suzhou Industrial Park, Jiangsu Province Applicant after: Suzhou kansel medical laboratory Co.,Ltd. Address before: Unit 306, B1 Floor, 218 Xinghu Street, Suzhou Industrial Park, Jiangsu Province Applicant before: SUZHOU CANCERCELL BIOTECHNOLOGY Co.,Ltd. |
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200327 |