CN109060488B - Tissue bluing liquid - Google Patents
Tissue bluing liquid Download PDFInfo
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- CN109060488B CN109060488B CN201810897090.7A CN201810897090A CN109060488B CN 109060488 B CN109060488 B CN 109060488B CN 201810897090 A CN201810897090 A CN 201810897090A CN 109060488 B CN109060488 B CN 109060488B
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- 239000007788 liquid Substances 0.000 title claims description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 29
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical class [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims abstract description 27
- 210000001519 tissue Anatomy 0.000 claims abstract description 26
- 238000010186 staining Methods 0.000 claims abstract description 10
- 210000001165 lymph node Anatomy 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 6
- 230000008569 process Effects 0.000 claims description 4
- 229960001484 edetic acid Drugs 0.000 abstract description 27
- 230000000694 effects Effects 0.000 abstract description 18
- 239000011259 mixed solution Substances 0.000 abstract description 18
- 239000000243 solution Substances 0.000 abstract description 17
- 210000002751 lymph Anatomy 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 abstract description 8
- 210000003855 cell nucleus Anatomy 0.000 abstract description 8
- 239000012670 alkaline solution Substances 0.000 abstract description 5
- 108010077544 Chromatin Proteins 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 4
- 210000003483 chromatin Anatomy 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 210000004940 nucleus Anatomy 0.000 abstract description 3
- 238000003908 quality control method Methods 0.000 abstract description 3
- 239000003086 colorant Substances 0.000 abstract description 2
- 238000011156 evaluation Methods 0.000 abstract description 2
- 239000002245 particle Substances 0.000 abstract description 2
- 238000007447 staining method Methods 0.000 abstract description 2
- 210000001280 germinal center Anatomy 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 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 description 12
- 210000004698 lymphocyte Anatomy 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 8
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 4
- 238000010827 pathological analysis Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- UKZQEOHHLOYJLY-UHFFFAOYSA-M ethyl eosin Chemical compound [K+].CCOC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 UKZQEOHHLOYJLY-UHFFFAOYSA-M 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910052808 lithium carbonate Inorganic materials 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 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 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- FUJCRWPEOMXPAD-UHFFFAOYSA-N lithium oxide Chemical compound [Li+].[Li+].[O-2] FUJCRWPEOMXPAD-UHFFFAOYSA-N 0.000 description 1
- 229910001947 lithium oxide Inorganic materials 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
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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
-
- 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
- G01N2001/302—Stain compositions
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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
In order to well perform the quality control between rooms of the conventional tissue slices, ensure the clarity of nuclear plasma of the conventional tissue slices and meet the requirements of excellent histological slice preparation. The method takes a mixed solution of saturated lithium carbonate and Ethylene Diamine Tetraacetic Acid (EDTA) as a tissue bluing solution, takes an appendix operation specimen with abundant cell mass as a research object, takes the staining effect of lymph nodes in the appendix as an evaluation standard, and researches the bluing effect of different alkaline solutions in hematoxylin-eosin staining method hematoxyli-eoitaig (HE) staining. The result proves that the mixed alkaline bluing solution has better effect than the pure solution, the bluing effect is best by the mixed solution of saturated lithium carbonate and EDTA, lymph nodules are obvious under the action of the mixed solution with the volume ratio of 1:4, the centers of the nodules can see lightly dyed germinal centers, lymph cell nuclei are blue-blue, the colors are bright, the nuclei and the plasma blue red are opposite, the contrast is good, the nuclear chromatin particles are clear, and the conventional excellent slicing standard is met.
Description
Technical Field
The invention provides a tissue bluing liquid used in the staining process by adopting a hematoxylin-eosin staining method, and the tissue bluing liquid is applied to the staining evaluation of lymph nodes in the appendix.
Background
Hematoxylin-eosin staining (he) is one of the most commonly used technical methods in conventional staining in pathology department, and hematoxylin in which an alkaline staining solution is used to stain chromatin and nucleic acid blue, which is in sharp contrast to the cytoplasm stained by acid dye eosin to distinguish the cell morphology in different tissue pathologies. In the practical operation process, hematoxylin is firstly differentiated by an acid solution to remove excessive combined dye and areas which are not to be colored, and then colored cells are blue in an alkaline solution to become clearer and more moderate.
The appendix has small and irregular lumen, abundant cell types, more goblet cells in the epithelium, and abundant lymphoid tissue in the lamina propria, and a large number of lymph nodules can continuously form layers and protrude into the submucosa. The lymph nodes are divided into primary lymph nodes and secondary lymph nodes, and the number, the form and the structure of the lymph nodes are dynamically changed under the stimulation of antigens. Lymphocytes vary in size, with many small lymphocytes. The small lymphocytes are similar to red cells and nuclein in size, one side of the small lymphocytes is always shallow, and chromatin is precise and blocky and is deeply colored; the cytoplasm was small, strongly basophilic and blue in color. Medium lymphocytes are smaller than neutrophils, have slightly lighter nuclear staining, more cytoplasmic smaller lymphocytes and bluish blue, and contain a small amount of azurophilic granules. Lymph nodules are important morphological markers reflecting the humoral immune response. The excellent tissue section staining can perfectly present lymph nodules.
Disclosure of Invention
Aiming at the problems, the invention takes the appendix operation specimen as a research object, researches the bluing effect and the optimal mixing ratio of different alkaline solutions in HE staining, meets the requirement of excellent conventional tissue sections, well controls the interventricular quality of the conventional tissue sections, perfectly presents the cell morphology of the tissues and improves the accuracy of pathological diagnosis.
The tissue bluing solution is a mixed solution of saturated lithium carbonate and EDTA.
The volume ratio of the saturated lithium carbonate to the EDTA mixed solution is 0.2-5: 1.
More preferably, the volume ratio of the saturated lithium carbonate to EDTA mixed solution is 1: 4.
Hematoxylin differentiated by hydrochloric acid and ethanol in the HE staining process is in a red ion state under an acidic condition and is in a blue ion state under an alkaline condition, after differentiation, the differentiation is stopped by washing with water to remove acid, and the cell nucleus stained by hematoxylin is blue in blue, namely bluish. Generally, the pathological department mostly uses tap water for immersion washing and bluing, but the quality of water in different places cannot meet the quality control requirement among rooms of the conventional HE tissue slices.
Most of the flowing tap water and part of the simple solution adopted by the invention can not blue the tissue cell nucleus, the contrast with the pulp is poor, the color of the nuclear pulp is similar, the region boundary is not obvious, and the excellent rate of the conventional section and the accuracy of pathological diagnosis are influenced. EDTA is an alkaline solution with the pH value of about 9.0, a saturated lithium carbonate aqueous solution is alkaline, after the two alkaline solutions are mixed according to different volumes, particularly, the bluing effect of a mixed solution (volume ratio is 1:4) of saturated lithium carbonate and EDTA is obviously better than that of a pure solution, but under the heating condition (about 50 ℃), lithium carbonate can be partially decomposed into lithium oxide and carbon dioxide, and the pH value of the mixed solution is directly influenced, so that the bluing effect is poor. The application of the saturated lithium carbonate and EDTA mixed solution (volume ratio of 1:4) to the blue returning solution in HE dyeing at room temperature has the advantages of convenience in operation, low cost, ideal effect and the like, and the commercialization of the saturated lithium carbonate and EDTA mixed solution (volume ratio of 1:4) is feasible.
The excellent conventional tissue slice can truly reflect the normal form and the abnormal shape of the cells of the tissue, the pathological change area and the cells are searched under the normal tissue form, the basis is provided for the diagnosis and classification of diseases, the accuracy of pathological diagnosis is improved, the intercellular quality control standard of the conventional tissue slice can be maximally achieved, and the pathological diagnosis level and the clinical comprehensive strength of a hospital can be comprehensively improved.
Drawings
Fig. 1 shows the bluing effect of different bluing liquids in HE dyeing at 200 magnifications, wherein a. is 0.3% ammonia water by mass fraction; B. saturated lithium carbonate; C. EDTA; D. running tap water; E. a saturated lithium carbonate and EDTA mixed solution (volume ratio is 4: 1); F. a mixed solution of saturated lithium carbonate and EDTA (volume ratio is 1: 1); G. saturated lithium carbonate and EDTA mixed solution (volume ratio 1: 4).
Detailed Description
Example 1
In the technical scheme of the application, 50 appendiceal surgery specimens are selected. The specimen pretreatment is consistent.
A full-automatic closed tissue dehydrator (Thermo, Excelsior AS), a tissue embedding machine (Changzhou Wei, BMJ-B), a paraffin slicer (Thermo, HM325), a floating machine (Changzhou Wei, PPJ-A), and an electric heating constant temperature drying oven (Shanghai leap, Hengzi).
Ethylenediaminetetraacetic acid (EDTA) was purchased from Dako corporation. TO, hematoxylin, lithium carbonate, alcohol-soluble eosin and hydrochloric acid alcohol solution are purchased from BASO company, and alcohol and ammonia water are purchased from Xiong chemical corporation
All sections in the experiment were 3 μm thick. Baking slices at 65 deg.C for 25min, dewaxing to water, staining with hematoxylin at room temperature for 15min, differentiating with 0.5% differentiation solution for 2s, returning blue to blue solution for 1min, staining with alcohol-soluble eosin for 2s, dehydrating, transparentizing, and sealing. The bluing solution respectively selects 0.3% ammonia water, saturated lithium carbonate, EDTA, flowing tap water, and a mixed solution of saturated lithium carbonate and EDTA (volume ratio is 1:1, 1:4, and 4: 1).
According to the technical scheme, under the same pretreatment, dyeing steps and dyeing conditions, aiming at 50 cases of appendiceal operation specimens, seven tissue bluing solutions are respectively selected, bluing effects and optimal mixing ratios of ammonia water with the mass fraction of 0.3%, saturated lithium carbonate, EDTA (ethylene diamine tetraacetic acid), flowing tap water and a mixed solution of saturated lithium carbonate and EDTA (the volume ratio is 1:1, 1:4 and 4:1) are compared, and experimental results obtained by bluing in the experiment are consistent.
The blue returning effect of the seven blue returning liquids in HE dyeing is shown in figure 1. The results show that seven kinds of bluing solutions have bluing effects in HE staining, but different bluing solutions have differences in tissue bluing effects, and the comparative analysis on bluing differences in HE staining indicates that (1) tissue sections with 0.3% ammonia water, flowing tap water, a mixed solution of saturated lithium carbonate and EDTA (volume ratio 4:1), and a mixed solution of saturated lithium carbonate and EDTA (volume ratio 1:1) (as shown in fig. 1A, 1D, 1E, and 1F) cannot present tissue structures, cannot achieve the bluing effect of cell nuclei, have purplish red color and poor contrast with plasma, cannot identify lymphocytes and lymph nodules, cannot achieve the mapping of nuclei and plasma blue red color, have clear nuclear plasma, and directly affect the yield and the diagnosis accuracy of tissue sections; (2) the bluing effect of the saturated lithium carbonate and the EDTA pure solution is slightly better (as shown in figures 1B and 1C), wherein the bluing effect of the EDTA pure solution is better than that of the saturated lithium carbonate, the lymphocyte blue stain is better compared with the pulp, but the secondary lymph nodule hair-growing center is not prominent; (3) the mixed solution of saturated lithium carbonate and EDTA in different volume ratios has different bluish colors of cell nuclei, and the cell nuclei of the lymphocytes are black-purple (as shown in FIG. 1E) under the action of the volume ratio of 1:4, purple-red (as shown in FIG. 1F) under the action of the volume ratio of 1:1, and the cell nuclei of the lymphocytes only show corresponding blue (as shown in FIG. 1G) under the condition of the volume ratio of 1: 4; (4) under the action of the same bluing time (1min), the seven bluing liquids are mixed with saturated lithium carbonate and EDTA (volume ratio 1:4) (as shown in figure 1G), so that the bluing effect is rapid, the bluing effect is obvious, the manual dyeing duration is shortened, the dyeing efficiency is improved, the tissue structure morphology can be perfectly presented, secondary lymph nodule hair growth centers with light dyeing in the center can be seen in the figure, peripheral lymph cell nuclei are bluish, the color is bright, the nuclei are in blue-blue with plasma red, the contrast is good, the chromatin particles are clear, and the conventional excellent slicing standard is met.
Claims (1)
1. The application of the tissue bluing liquid in the hematoxylin-eosin staining process of appendiceal lymph node is characterized in that the bluing liquid is a mixed liquid of saturated lithium carbonate and EDTA, and the volume ratio of the saturated lithium carbonate to the EDTA is 1: 4.
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| CN201810897090.7A CN109060488B (en) | 2018-08-08 | 2018-08-08 | Tissue bluing liquid |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694732A (en) * | 2013-12-31 | 2014-04-02 | 江苏省原子医学研究所 | Hematoxylin staining solution and HE (hematoxylin eosin) staining solution containing hematoxylin staining solution |
| CN104744967A (en) * | 2013-12-31 | 2015-07-01 | 江苏省原子医学研究所 | Eosin staining solution and HE staining solution containing same |
| CN105628913A (en) * | 2016-01-13 | 2016-06-01 | 深圳中山生殖与遗传研究所 | Kit for evaluating endometrial receptivity and using method thereof |
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- 2018-08-08 CN CN201810897090.7A patent/CN109060488B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694732A (en) * | 2013-12-31 | 2014-04-02 | 江苏省原子医学研究所 | Hematoxylin staining solution and HE (hematoxylin eosin) staining solution containing hematoxylin staining solution |
| CN104744967A (en) * | 2013-12-31 | 2015-07-01 | 江苏省原子医学研究所 | Eosin staining solution and HE staining solution containing same |
| CN105628913A (en) * | 2016-01-13 | 2016-06-01 | 深圳中山生殖与遗传研究所 | Kit for evaluating endometrial receptivity and using method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 结石形成中骨桥蛋白与CD44的作用研究;于德君等;《大连医科大学学报》;20111231;第33卷(第6期);第537页第1.4节 * |
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