CN102175496A - Formaldehyde-free fixative for tissue sample - Google Patents
Formaldehyde-free fixative for tissue sample Download PDFInfo
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- CN102175496A CN102175496A CN 201110023748 CN201110023748A CN102175496A CN 102175496 A CN102175496 A CN 102175496A CN 201110023748 CN201110023748 CN 201110023748 CN 201110023748 A CN201110023748 A CN 201110023748A CN 102175496 A CN102175496 A CN 102175496A
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Abstract
The invention provides a formaldehyde-free fixative for a tissue sample, which comprises the following ingredients in parts by volume: 710-790 parts of ethanol, 20-40 parts of glacial acetic acid, 10-30 parts of acetone, 40-60 parts of polyhydric alcohol, 40-60 parts of trichloroacetic acid (TDA), 40-60 parts of surface active agent and 40-60 parts of solubilizing agent. The formaldehyde-free fixative is a nontoxic and innocuous chemical reagent; after being proved by cellular morphological experiments, immunohistochemical experiments, molecular pathological experiments and nucleic acid extraction experiments, the formaldehyde-free fixative has relatively comprehensive fixing effect, can replace the formaldehyde and be used in related pathological experiments, thereby fixing the tissue sample.
Description
Technical field
The present invention relates to external diagnosis reagent, be specifically related to a kind of environment-friendly formaldehyde-free tissue specimen immobile liquid and application thereof, belong to pathology experimental technique field.
Background technology
The history in existing more than 150 year of histopathologic birth, in all hospitals, the Pathologic specimen of excision and various biopsy tissue are made histopathologic diagnosis by hospital pathology department to the operation tissue samples.
Routine paraffin wax embedding HE stained preparation is current basic fundamental in order to handling tissue samples, and it is the methodological basis of pathological diagnosis, also is the evaluation reference standard of many new methods, new technology quality.Traditional method is that tissue samples is placed in the cylinder for the treatment of fluid, pours or call in different disposal liquid successively into, and sample soaks different time successively in accordance with regulations and finishes processing section, dyeing, mounting etc. in each reagent, and whole process needs more than 30 step.Whole process is used formaldehyde, dimethylbenzene, mercury oxide, the toxic chemical reagent of ammoniacal liquor in a large number, and the pathology technique personnel place oneself in the midst of poisonous environment for a long time, and the reagent emission of serious pollution of environment.And used reagent tolerance is poor, complicated operation, and reagent consumption is big, pathology film-making high-quality sheet low (whole nation statistics is 40～70%), influence diagnosis.The exhausted simultaneously big hospital of counting joins reagent voluntarily, lacks unified standard.Therefore the standardized production of pathology industry organization sample disposal liquid articles for use, do not have poison, the socialization supply and demand, be the inexorable trend of industry development.
For example application number is that the patented claim of 200810136961.X discloses a kind of biological organization sample routine paraffin wax flaking HE dyeing non-xylol whole-course treating fluid.Yet this treating fluid based on traditional fixation of tissue has still adopted the murder by poisoning reagent formaldehyde of serious environment pollution.Use the formaldehyde-formalin of variable concentrations, fixing human and animal vegetable tissue sample carry out Human pathology and the existing history more than 160 years of animals and plants pathological research.Formaldehyde is that the poisonous and harmful chemicals of generally acknowledging has potential carcinogenicity, and it is a kind of simple fixative, and its fixed mechanism is by the reaction of the hinge between the protein subunit amino acid molecular, forms methylene bridge and finishes fixation.Yet phenomenons such as sclerosis can appear shrinking in the hinge reaction, preserve not good to histiocytic antigen molecule structure, enzyme, nucleic acid molecules.Therefore, the substitute instead of formaldehyde of research and development formaldehyde is used for pathology experiments and has great importance.
Summary of the invention
The object of the present invention is to provide a kind of formaldehydeless tissue specimen immobile liquid, can satisfy routine pathology diagnosis, immunohistochemical diagnosis technology, diagnosis of molecular biology technical requirement, the tissue effect that handles is better than conventional art, tests with the pathology that complete instead of formaldehyde is applied to be correlated with.The concentration of each reagent of the present invention is mass percentage concentration.
In order to reach purpose of the present invention, adopt following technical scheme:
The invention provides a kind of formaldehydeless tissue specimen immobile liquid, it comprises the component of following volume parts:
Ethanol 710-790 part, glacial acetic acid 20-40 part, acetone 10-30 part, polyvalent alcohol 40-60 part, trichloroacetic acid 40-60 part, surfactant 40-60 part, solubilizer 40-60 part.
In the preferred embodiment of the present invention, described formaldehydeless tissue specimen immobile liquid comprises the component of following volume parts: 750 parts of ethanol, 30 parts of glacial acetic acids, 20 parts in acetone, 50 parts of polyvalent alcohols, 50 parts of trichloroacetic acids, 50 parts in surfactant, 50 parts of solubilizer.
Wherein said ethanol is concentration 75-95% ethanol, is preferably 95% ethanol.
Wherein said polyvalent alcohol is selected from diglycol, triethylene glycol, polyglycol, is preferably diglycol.
Wherein said surfactant is selected from quaternary ammonium salt surface active agent or fluorine class surfactant, preferably from octyl group-decyl-alkyl dimethyl ammonium chloride, Quaternium 24, DDAC, didecyl dimethyl ammonium bromide, more preferably octyl group-decyl-alkyl dimethyl ammonium chloride.Described surfactant concentrations is 0.5-2%, preferred 1%.
Described solubilizer is selected from 3-methoxyl-3-methyl butanol or 3-methoxyl-3-methyl isophthalic acid-butyl acetate, preferred 3-methoxyl-3-methyl butanol; Its concentration is 0.5-2%, preferred 1%.
Because the present invention does not use formaldehyde, therefore both avoided the toxic action of formaldehyde, eliminated the covering of formaldehyde again to antigenic substance, can be good at preserving antigen.And the present invention use polyvalent alcohol for example diglycol or polyglycol promote the osmosis of molecule to finish fixation procedure to promote the cell protein precipitating action, the phenomenons such as contraction sclerosis that this process has suppressed the hinge reaction help the preservation of histocyte form.
The preparation of formaldehydeless tissue specimen immobile liquid provided by the invention is that each component is got final product in described ratio mixing at normal temperatures.
Formaldehydeless tissue specimen immobile liquid provided by the invention adopts the composite parts prescription, its whole components are nontoxic chemical reagent, experimental results show that through cytomorphology experiment, immunohistochemical assay, molecular pathology experiment and nucleic acid extraction, no aldehyde immobile liquid has comparatively comprehensively fixed effect, the pathology experiment that fully can instead of formaldehyde is applied to be correlated with is to finish the fixation to tissue specimen.
Description of drawings
The rabbit renal tissue after stain chromatic graph (LEICA DM4000B cinephotomicrography 200 times) of Fig. 1 for adopting formaldehydeless tissue specimen immobile liquid of the present invention to handle.
The people renal tissue after stain chromatic graph (LEICA DM4000B cinephotomicrography 200 times) of Fig. 2 for adopting formaldehydeless tissue specimen immobile liquid of the present invention to handle.
The adenocarcinoma of endometrium estrogen receptor immunohistochemical staining figure (LEICA DM4000B cinephotomicrography 200 times) of Fig. 3 A for adopting formaldehydeless tissue specimen immobile liquid of the present invention to handle.
The endometrial hyperplasia progesterone receptor immunohistochemical staining figure (LEICA DM4000B cinephotomicrography 200 times) of Fig. 3 B for adopting formaldehydeless tissue specimen immobile liquid of the present invention to handle.
The human breast carcinoma c-erbB-2 gene expression protein immunization histochemical stain figure (LEICA DM4000B cinephotomicrography 200 times) of Fig. 4 for adopting formaldehydeless tissue specimen immobile liquid of the present invention to handle.
Embodiment
The invention will be further described below in conjunction with embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment 1
95% ethanol--------------------------------750ml
Glacial acetic acid------------------------------------30ml
Acetone--------------------------------------20ml
Diglycol------------------------------------50ml
Trichloroacetic acid (2%)-----------------------------50ml
Octyl group-decyl-alkyl dimethyl ammonium chloride (1%)---50ml
3-methoxyl-3-methyl butanol (1%)--------50ml
Said components is promptly miscible at normal temperatures.
Embodiment 2
90% ethanol------------------------------710ml
Glacial acetic acid---------------------------------20ml
Acetone-----------------------------------30ml
Diglycol---------------------------------60ml
Trichloroacetic acid (2%)--------------------------60ml
Quaternium 24 (0.5%)---60ml
3-methoxyl-3-methyl butanol (1%)---------60ml
Said components is promptly miscible at normal temperatures.
Embodiment 3
75% ethanol------------------------------790ml
Glacial acetic acid---------------------------------40ml
Acetone------------------------------------10ml
Polyglycol--------------------------------40ml
Trichloroacetic acid (2%)--------------------------40ml
DDAC (2%)------40ml
3-methoxyl-3-methyl butanol (0.5%)------40ml
Said components is promptly miscible at normal temperatures.
Embodiment 4
80% ethanol------------------------------750ml
Glacial acetic acid-------------------------------30ml
Acetone---------------------------------20ml
Triethylene glycol-------------------------------50ml
Trichloroacetic acid (2%)-----------------------50ml
Didecyl dimethyl ammonium bromide (1%)---------50ml
3-methoxyl-3-methyl isophthalic acid-butyl acetate (2%)--50ml
Said components is promptly miscible at normal temperatures.
Test effect
Adopt formaldehydeless tissue specimen immobile liquid provided by the invention, on sample process, stopped conventional art to tissue samples handle often occur make tissue samples contract, firmly, split, crisp phenomenon, make the more approaching primary form of cell and institutional framework, fixing intact to institutional framework, dehydration thoroughly.
Aspect molecular biology experiment and research, experimental results show that the rabbit renal tissue poststaining of the formaldehydeless tissue specimen immobile liquid processing that employing embodiment 1 provides, see Fig. 1 (200 times of LEICA DM4000B cinephotomicrographies), display organization is fixed well, clear in structure, nucleus-slurry-membrane structure are intact.。
People's renal tissue poststaining that the formaldehydeless tissue specimen immobile liquid that adopts embodiment 2 to provide is handled is seen Fig. 2 (200 times of LEICA DM4000B cinephotomicrographies), and display organization is fixed well, fiber (basket) clear in structure in glomerular basement membrane (deceiving) and the crescent.
The adenocarcinoma of endometrium estrogen receptor immunohistochemical staining that the formaldehydeless tissue specimen immobile liquid that adopts embodiment 3 to provide is handled is seen Fig. 3 A, endometrial hyperplasia progesterone receptor immunohistochemical staining is seen Fig. 3 B, and Fig. 4 (being 200 times of LEICADM4000B cinephotomicrographies) is seen in the histochemical stain of human breast carcinoma c-erbB-2 gene expression protein immunization, the immunogenicity of display organization cell is preserved good, the distribution of demonstration antigen that can be good and correct location.
The above only is preferred embodiment of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive.Those skilled in the art is understood, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even equivalence, but all will fall within the scope of protection of the present invention.
Claims (8)
1. formaldehydeless tissue specimen immobile liquid, it comprises the component of following volume parts: ethanol 710-790 part, glacial acetic acid 20-40 part, acetone 10-30 part, polyvalent alcohol 40-60 part, trichloroacetic acid 40-60 part, surfactant 40-60 part, solubilizer 40-60 part.
2. formaldehydeless tissue specimen immobile liquid according to claim 1, it comprises the component of following volume parts: 750 parts of ethanol, 30 parts of glacial acetic acids, 20 parts in acetone, 50 parts of polyvalent alcohols, 50 parts of trichloroacetic acids, 50 parts in surfactant, 50 parts of solubilizer.
3. formaldehydeless tissue specimen immobile liquid according to claim 1 and 2, wherein said ethanol is concentration 75-95% ethanol, is preferably 95% ethanol.
4. formaldehydeless tissue specimen immobile liquid according to claim 1 and 2, wherein said polyvalent alcohol is selected from diglycol, triethylene glycol, polyglycol, is preferably diglycol.
5. formaldehydeless tissue specimen immobile liquid according to claim 1 and 2, wherein said surfactant are selected from quaternary ammonium salt surface active agent or fluorine class surfactant, and its concentration is 0.5-2%, and preferred 1%.
6. formaldehydeless tissue specimen immobile liquid according to claim 5, wherein said quaternary ammonium salt surface active agent is to be selected from octyl group-decyl-alkyl dimethyl ammonium chloride, Quaternium 24, DDAC, didecyl dimethyl ammonium bromide, preferred octyl group-decyl-alkyl dimethyl ammonium chloride.
7. formaldehydeless tissue specimen immobile liquid according to claim 1 and 2, wherein said solubilizer are selected from 3-methoxyl-3-methyl butanol or 3-methoxyl-3-methyl isophthalic acid-butyl acetate, preferred 3-methoxyl-3-methyl butanol; Its concentration is 0.5-2%, preferred 1%.
8. the application of each described formaldehydeless tissue specimen immobile liquid of claim 1-7 is to be used for Histopathology and animal or plant specimen making, is used for routine pathology diagnosis, immunohistochemical diagnosis technology, diagnosis of molecular biology technology.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663647A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Heart tissue fixing agent as well as preparation method and application of heart tissue fixing agent |
| CN104663646A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Embryonic tissue fixative and its preparation method and use |
| CN105842033A (en) * | 2016-05-13 | 2016-08-10 | 北京九州柏林生物科技有限公司 | Formaldehyde-free tissue specimen stationary liquid |
| CN106323704A (en) * | 2015-07-01 | 2017-01-11 | 深圳市廖氏原生物医学科技有限公司 | Environmentally friendly tissue fixative solution and production method thereof |
| CN106706395A (en) * | 2016-12-08 | 2017-05-24 | 武汉宏兹生物技术有限公司 | Novel environment-friendly stationary liquid |
| CN108967414A (en) * | 2017-05-31 | 2018-12-11 | 王虎 | USBU is without formalin fixer and its preparation method and application |
| CN109805001A (en) * | 2017-11-22 | 2019-05-28 | 哈尔滨格林标本技术开发有限公司 | Formaldehydeless tissue Fixed and Antiseptic liquid of one kind and preparation method thereof |
| CN114279801A (en) * | 2021-12-24 | 2022-04-05 | 哈尔滨东尧科技有限公司 | Formaldehyde-free fixing solution for fixing various fresh tissues and fixing method |
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| EP0775439A1 (en) * | 1995-03-09 | 1997-05-28 | Universidad Complutense De Madrid | Compositions containing dialkyl (c1-c6)-ketone peroxide for the preservation of organic tissues, and application of said compositions to the preservation and anatomical preparation of organic tissues of animal or human origin |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663647A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Heart tissue fixing agent as well as preparation method and application of heart tissue fixing agent |
| CN104663646A (en) * | 2013-11-27 | 2015-06-03 | 深圳先进技术研究院 | Embryonic tissue fixative and its preparation method and use |
| CN106323704A (en) * | 2015-07-01 | 2017-01-11 | 深圳市廖氏原生物医学科技有限公司 | Environmentally friendly tissue fixative solution and production method thereof |
| CN105842033A (en) * | 2016-05-13 | 2016-08-10 | 北京九州柏林生物科技有限公司 | Formaldehyde-free tissue specimen stationary liquid |
| CN106706395A (en) * | 2016-12-08 | 2017-05-24 | 武汉宏兹生物技术有限公司 | Novel environment-friendly stationary liquid |
| CN108967414A (en) * | 2017-05-31 | 2018-12-11 | 王虎 | USBU is without formalin fixer and its preparation method and application |
| CN109805001A (en) * | 2017-11-22 | 2019-05-28 | 哈尔滨格林标本技术开发有限公司 | Formaldehydeless tissue Fixed and Antiseptic liquid of one kind and preparation method thereof |
| CN109805001B (en) * | 2017-11-22 | 2021-01-01 | 哈尔滨格林标本技术开发有限公司 | Formaldehyde-free tissue fixation preservative solution and preparation method thereof |
| CN114279801A (en) * | 2021-12-24 | 2022-04-05 | 哈尔滨东尧科技有限公司 | Formaldehyde-free fixing solution for fixing various fresh tissues and fixing method |
| CN114279801B (en) * | 2021-12-24 | 2024-03-29 | 哈尔滨东尧科技有限公司 | Formaldehyde-free fixing liquid and fixing method for fixing various fresh tissues |
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