CN114279801B - Formaldehyde-free fixing liquid and fixing method for fixing various fresh tissues - Google Patents
Formaldehyde-free fixing liquid and fixing method for fixing various fresh tissues Download PDFInfo
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Abstract
The invention discloses formaldehyde-free fixing liquid capable of being used for fixing various fresh tissues, which comprises liquid A and liquid B, and sequentially carries out first fixing and second fixing on the fresh tissues, so that various substances in the various tissues can be effectively fixed, and the fixed tissues are not obvious in expansion and contraction, so that the tissues keep the morphological structure in living state as much as possible, and the subsequent slicing and pathological examination are facilitated.
Description
Technical Field
The invention belongs to the technical field of medical histopathology, and particularly relates to formaldehyde-free fixing liquid and a fixing method for fixing various fresh tissues.
Background
In histopathology, the collected fresh tissue needs to be fixed properly and effectively in time to prevent autolysis and putrefaction of the tissue, so that components such as proteins, fats, sugar, enzymes and the like in cells are precipitated or coagulated, the tissues and the cells keep morphological structures in living states as much as possible, and subsequent slicing and other works can be carried out smoothly.
The fixing solution is a common tissue fixing agent, the traditional tissue fixing solution is formalin (formaldehyde aqueous solution), the problems of easy volatilization and high toxicity of formaldehyde exist, the environment is easy to be polluted in the use process, and the body of an operator is damaged. Meanwhile, the human tissues are various in types, different in tissue structures and properties at different parts, and along with the development of immunohistochemistry and molecular biology, the fixing solution is required to have diversity. However, if one fixing liquid is used for each human tissue, a large amount of fixing liquids need to be prepared and marked, respectively, with a great amount of work.
Ethanol is used as a tissue fixative, which has both fixation and dehydration effects on tissue cells, if ethanol with high concentration is directly used as the fixative, the surface of a biological material is rapidly dehydrated to form a layer of structure similar to a protective film, fixation and dehydration inside the tissue are affected, thus the concentration of the ethanol is required to have gradient (the concentration is required to be high to low), the concentration gradient adopted in the prior art is generally 50% -70% -80% -95% -absolute ethanol, the span change among gradients of the ethanol concentration is larger, the fixation and dehydration effects are poor, if ethanol solutions with smaller spans among gradients are required to be used, a plurality of groups of ethanol solutions with different concentrations are required to be prepared, frequent liquid exchange is required during fixation, the preparation, fixation and dehydration processes are complex, and the fixation mode in the prior art is an open treatment mode, so that sample pollution is easy to cause when the fixation liquid is required to be replaced for a plurality of times.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide formaldehyde-free fixing liquid which can be used for fixing various fresh tissues, the fixing liquid consists of various fixing liquid components, the fresh tissues can be sequentially fixed, various substances in various tissues can be effectively fixed, and the expansion and contraction of the fixed tissues are not obvious, so that the tissues keep the morphological structure in living state as much as possible, and the subsequent slicing and pathological examination are facilitated.
The aim of the invention is realized by the following technical scheme:
a formaldehyde-free fixative solution for fixation of multiple fresh tissues, comprising a solution A and a solution B;
the solution A comprises picric acid and glacial acetic acid;
the liquid B is prepared from the following raw materials: glutaraldehyde, ethanol.
Preferably, the liquid B comprises liquid B1, liquid B2 and liquid B3;
the B1 liquid is prepared from the following raw materials in parts by weight: 10 parts of glutaraldehyde, 50 parts of ethanol and 40 parts of phosphate buffer;
the raw materials for preparing the B2 solution also comprise phosphate buffer solution;
the B2 liquid is prepared from the following raw materials in parts by weight: 10 parts of glutaraldehyde, 70 parts of ethanol and 20 parts of phosphate buffer solution;
the B3 liquid is prepared from the following raw materials in parts by weight: glutaraldehyde 5 parts and ethanol 95 parts.
The glutaraldehyde is glutaraldehyde water solution, and the mass percentage of glutaraldehyde in the glutaraldehyde water solution is 24% -26%;
the concentration of the phosphate buffer solution is 0.2mol/L, and the pH value is 7.4;
the ethanol is absolute ethanol.
Preferably, the picric acid is a picric acid saturated solution, and the mass percentage of the picric acid saturated solution to the glacial acetic acid is 15:1, a step of;
the amount of the picric acid saturated solution is based on the condition that the picric acid saturated solution can be dissolved in water and form a picric acid saturated aqueous solution.
A method of preparing a formaldehyde-free fixative solution useful for fixation of multiple fresh tissues comprising the steps of:
step one, preparing a solution A:
taking picric acid and glacial acetic acid according to mass percent, and uniformly stirring and mixing to obtain solution A;
step two, preparing a solution B:
preparing a B1 solution: taking glutaraldehyde, ethanol and phosphate buffer solution according to parts by weight, and uniformly stirring and mixing to obtain solution B1;
preparing a B2 solution: taking glutaraldehyde, ethanol and phosphate buffer solution according to parts by weight, stirring and mixing uniformly to prepare solution B2;
preparing a solution B3: and (3) taking glutaraldehyde and ethanol according to the parts by weight, and stirring and uniformly mixing to obtain the solution B3.
Preferably, the method for fixing fresh tissue using the fixing solution comprises the steps of:
s1, placing fresh tissues in the solution A, and performing first fixation for 3-5 hours;
when the first fixation is carried out, the volume ratio of the liquid A to the tissue to be fixed is 10-30:1, preferably 20-30:1, a step of;
s2, after the first fixation is completed, washing the tissue by using PBS buffer solution;
s3, placing the cleaned tissue in a fixing chamber, adding the liquid B, and performing second fixing;
and in the second fixation, the volume ratio of the liquid B to the tissue to be fixed is 10-30:1, preferably 20-30:1.
preferably, the step of adding liquid B includes replacement of the partially used liquid.
Preferably, the order of adding the solutions in the solution B in the step S3 is as follows: firstly adding the B1 solution, then adding the B2 solution and finally adding the B3 solution.
Preferably, in the second fixing process in S3, the following three liquid changing modes are sequentially performed:
in the first liquid exchange mode, when the B2 liquid is required to be added into the fixed chamber, if the liquid in the current fixed chamber is the used B1 liquid, discharging part of the liquid in the current fixed chamber and adding the B2 liquid, and performing the first liquid exchange;
in the second liquid changing mode, when the B2 liquid is required to be added into the fixed chamber, if the liquid in the current fixed chamber comprises the used B2 liquid, discharging part of the liquid in the current fixed chamber, adding the B2 liquid, and performing the second liquid changing;
and in the third liquid exchange mode, when the B3 liquid is required to be added into the fixed chamber, discharging part of the liquid in the current fixed chamber, adding the B3 liquid, and carrying out the third liquid exchange.
The mass percentage of the ethanol in the liquid in the fixed chamber is gradually increased along with the increase of the adding times of the B2 liquid and the B3 liquid.
Preferably, a linkage chamber is arranged above the fixed chamber, a movable piston is arranged between the linkage chamber and the fixed chamber, and can move up and down between the linkage chamber and the fixed chamber in a sealing manner when the movable piston is closed, and the movable piston is used for changing the volumes of the fixed chamber and the linkage chamber;
the replacement of the partially used liquid comprises the following steps:
when it is desired to replace a portion of the liquid in the stationary chamber,
s101, filling new liquid into the linkage chamber, moving the piston to descend in a force accumulation mode, driving the linkage assembly to move to the opening of the liquid discharge hole, and discharging part of used liquid in the fixed chamber from the liquid discharge hole in the opening state;
s102, after the movable piston descends to a set position, driving the linkage assembly to move to the liquid discharge hole for reclosing;
s103, opening the movable piston to expose an empty chamber of the movable piston, communicating a fixed chamber, a linkage chamber and the empty chamber, filling new liquid into the empty chamber, releasing the accumulated force, keeping the liquid discharge hole closed while resetting the movable piston until the movable piston is reset, closing the movable piston, and sealing and separating the linkage chamber and the fixed chamber again to finish the replacement of part of used liquid.
Preferably, the linkage assembly comprises a linkage bar tooth and a transmission ratchet; the upper end of the linkage bar-shaped tooth is in running fit with the movable piston; the linkage bar-shaped teeth are in unidirectional linkage with the transmission ratchet teeth;
the up-and-down reciprocating motion of the linked bar-shaped teeth comprises the following steps:
step one, driving ratchets are contacted when the linked bar-shaped teeth move downwards to drive the driving ratchets to rotate in a force storage mode so as to open the liquid discharge hole;
step two, when the linked bar-shaped teeth continue to move downwards to be separated from the transmission ratchet, the transmission ratchet releases the power to reset and rotate, and the liquid discharge hole is closed;
and thirdly, in the upward resetting process of the linked bar-shaped teeth, the transmission ratchet is blocked by one way to limit the reverse rotation of the transmission ratchet, and the linked bar-shaped teeth overcome the blocking of the transmission ratchet in a deflection mode to reset upward.
The efficacy of the invention is based on:
picric acid: is an acidic fixing agent, can produce precipitation effect on all proteins, and has obvious shrinkage effect;
glacial acetic acid: the nuclear protein can be precipitated, and the chromosome structure can be well maintained;
glutaraldehyde: is an aldehyde with double functions, has better fixing function on glycogen, glycoprotein, microtubule endoplasmic reticulum, cell matrix and the like, and can well preserve tissues;
ethanol: is a lipid solvent capable of dissolving fat and lipoid, and can harden tissue, deform cell nucleus, shrink cytoplasm, slowly denature protein and precipitate glycogen after long-term soaking.
Compared with the prior art, the invention has the following beneficial effects:
the fixing liquid of the invention does not contain formaldehyde, avoids the pollution of formaldehyde to the air environment and the health threat of operators and related personnel in the using process, has much smaller stimulation of glutaraldehyde than formaldehyde under the same condition, and has higher environmental protection and safety. Firstly, preparing a solution A by picric acid and glacial acetic acid, and performing first fixing treatment on fresh tissues; the glacial acetic acid has a strong penetrating effect, so that the tissue can be fixed in a short time, and then glutaraldehyde and ethanol are used for further fixing the tissue, so that the defect of weak penetrating power of glutaraldehyde is effectively overcome, the tissue is quickly fixed, the fixed tissue is not obvious in expansion and contraction, the tissue maintains the morphological structure in a living state as much as possible, and the subsequent slicing and pathological examination are facilitated.
The tissue structure can be well preserved by the combined use of the picric acid and the glacial acetic acid, the picric acid can have precipitation effect on all proteins, but has contraction effect on tissues, and the glacial acetic acid can resist excessive contraction of tissues caused by other fixatives such as the picric acid while rapidly precipitating nucleoprotein and better maintaining the chromosome structure, and the picric acid can also soften the tissues, prevent the tissues from being excessively hardened and is unfavorable for subsequent slicing;
the glutaraldehyde and the ethanol are used for further fixing the tissue, the glutaraldehyde has better fixing effects on glycogen, glycoprotein, microtubule endoplasmic reticulum, cell matrix and the like, the ethanol can precipitate albumin, globulin and nucleoprotein, the ethanol has fixing effects on tissue cells and has dehydration effects, the ethanol with the concentration from low to high is used together with the glutaraldehyde, the tissue is fixed, the continuous gradient dehydration of the tissue is realized, the condition that the surface of the biological material is rapidly dehydrated by high-concentration ethanol to form a layer of structure similar to a protective film is avoided, the dehydration of the internal material is influenced, the tissue subjected to the second fixing treatment can be subjected to subsequent treatments such as transparency, embedding, slicing and the like only by the final dehydration treatment of absolute ethanol, the synchronous tissue fixing and tissue dehydration are realized, and the efficiency of pathological examination is effectively improved;
in addition, the picric acid in the A liquid used for the first fixation can cause the tissue to yellow, and the problem can be effectively solved after the treatment of the second fixation ethanol;
according to the invention, the mass percentage of the ethanol in the liquid B is from low to high gradient difference from the liquid B1 (the mass percentage of the ethanol is 50%), the liquid B2 (the mass percentage of the ethanol is 70%) and the liquid B3 (the mass percentage of the ethanol is 95%), the gradient span of the mass percentage of the ethanol in the liquid in the fixing chamber is reduced after the treatment of the liquid used by the replacement part, the gradient slow fixing and dehydration of the ethanol to tissues can be effectively realized, and the dehydration effect is good.
Drawings
FIG. 1 is a flow chart of a preparation process of tissue fluid in the invention;
FIG. 2 is a flow chart of the present invention for securing fresh tissue using interstitial fluid;
FIG. 3 is a schematic view of the overall internal structure of the tissue fixation device of the present invention;
FIG. 4 is a schematic view of the overall planar internal structure of the tissue fixation device of the present invention;
FIG. 5 is a second perspective view of the overall internal structure of the tissue fixation device of the present invention;
FIG. 6 is a schematic view of a partially exploded view of a moving piston of a tissue fixation device according to the present invention;
FIG. 7 is a schematic illustration of a portion of a linkage assembly of the tissue fixation device of the present invention;
FIG. 8 is a second schematic view of a portion of a linkage assembly of the tissue fixation device of the present invention;
FIG. 9 is a schematic view of a partial enlarged structure of the present invention at A of FIG. 8;
FIG. 10 is a schematic view of the structure of the driving rack and the driving gear of the tissue fixation device of the present invention.
In the figure: 1. a liquid tank; 11. a stationary chamber; 111. a guide rod; 12. a linkage chamber; 121. a water inlet pipe; 122. a control valve; 123. a baffle; 13. a pressure relief wall; 14. a pH detector; 2. moving the piston; 21. a cavity; 22. an opening; 23. a moving frame; 231. an electric push rod; 24. a hollow chamber; 25. a support rod; 3. a reset assembly; 31. a limiting cylinder; 32. a compression spring; 4. a liquid discharge hole; 41. a fixed rod; 5. a linkage assembly; 51. linkage bar-shaped teeth; 511. wedge-shaped protruding blocks; 52. a linkage gear set; 521. a driving ratchet; 522. a drive gear; 523. a sector seal block; 524. a fan-shaped through hole; 525. a torsion spring; 526. a fixing ring; 527. a transmission gear.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.
Example 1
Preparation of fixative solutions for fixation of various fresh tissues:
weighing the raw materials:
the preparation method comprises the following steps of (1) preparing the raw material of the solution A, and weighing the raw material according to mass percent: 15:1, picric acid saturated solution and glacial acetic acid;
raw materials for preparing the solution B:
the raw materials for preparing the B1 liquid are weighed according to the parts by weight: 10 parts of glutaraldehyde aqueous solution with the mass percentage content of 25% (namely, the mass percentage of glutaraldehyde in the glutaraldehyde aqueous solution is 25%), 40 parts of absolute ethyl alcohol and 50 parts of phosphoric acid buffer solution;
the raw materials for preparing the B2 liquid are weighed according to the parts by weight: 10 parts of glutaraldehyde aqueous solution with the mass percentage content of 25% (namely, the mass percentage of glutaraldehyde in the glutaraldehyde aqueous solution is 25%), 70 parts of absolute ethyl alcohol and 20 parts of phosphoric acid buffer solution;
the raw materials for preparing the B3 solution are weighed according to the parts by weight: 5 parts of glutaraldehyde aqueous solution with the mass percentage content of 25% (namely, the mass percentage of glutaraldehyde in the glutaraldehyde aqueous solution is 25%) and 95 parts of absolute ethyl alcohol;
the method comprises the following steps:
s1, preparing a solution A: mixing the picric acid saturated solution and glacial acetic acid uniformly to obtain solution A;
s2, preparing a solution B:
preparing a B1 solution: stirring and mixing glutaraldehyde water solution, absolute ethyl alcohol and phosphate buffer solution uniformly to obtain solution B1;
preparing a B2 solution: stirring and mixing glutaraldehyde water solution, absolute ethyl alcohol and phosphate buffer solution uniformly to obtain solution B2;
preparing a solution B3: and (3) uniformly stirring and mixing the glutaraldehyde aqueous solution and absolute ethyl alcohol to obtain the solution B3.
Example 2
The interstitial fluid prepared in example 1 was used for fixation of fresh tissues of the breast, comprising the following steps:
s1, placing fresh tissues of the mammary gland into A liquid, wherein the volume ratio of the A liquid to the fresh tissues of the mammary gland is 20:1, performing first fixing, and performing fixing treatment for 3 hours;
s2, after the first fixation is completed, washing breast tissues by using PBS buffer solution;
s3, placing the cleaned breast tissue in a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh tissue of the breast is 20:1, a step of;
the method comprises the following steps: firstly, filling B1 liquid into a fixing chamber in which the washed mammary tissue is placed for fixing;
after 3 hours of fixing treatment, injecting new B2 liquid to replace the liquid after partial use, wherein the liquid after partial use is replaced by injecting new B2 liquid once every 1 hour of fixing treatment, and the total replacement is 5 times;
after the 5 th time of filling new B2 liquid to replace the liquid after the partial use, fixing the liquid for 1 hour, filling new B3 liquid to replace the liquid after the partial use, and filling new B3 liquid to replace the liquid after the partial use once every 0.5 hour of fixing the liquid for 5 times.
The total fixation time of the first fixation and the second fixation was 13.5 hours.
Example 3
The interstitial fluid prepared in example 1 was used for fixation of fresh tissue of thyroid gland, comprising the following steps:
s1, placing a fresh thyroid group in a solution A, wherein the volume ratio of the solution A to the fresh thyroid tissue is 20:1, performing first fixing, and performing fixing treatment for 3 hours;
s2, after the first fixation is completed, washing thyroid tissues with PBS buffer solution;
s3, placing the cleaned thyroid tissue in a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh thyroid tissue is 20:1, a step of;
the method comprises the following steps: firstly, filling the fixing chamber with the cleaned thyroid tissue with B1 liquid for fixing;
after 3 hours of fixing treatment, injecting new B2 liquid to replace the liquid after partial use, wherein the liquid after partial use is replaced by injecting new B2 liquid once every 1 hour of fixing treatment, and the total replacement is 5 times;
after the 5 th time of replacement of the partially used liquid with the new B2 liquid, fixing treatment is performed for 1 hour, the new B3 liquid is injected to replace the partially used liquid, and every fixing treatment is performed for 1 hour, the new B3 liquid is injected to replace the partially used liquid for 5 times.
The total fixation time of the first fixation and the second fixation was 16 hours.
Example 4
The interstitial fluid prepared in example 1 was used for fixation of fresh tissues of ovaries, comprising the following steps:
s1, placing fresh tissues of ovaries in a solution A, wherein the volume ratio of the solution A to the fresh tissues of ovaries is 20:1, performing first fixing, and performing fixing treatment for 3 hours;
s2, after the first fixation is completed, cleaning the ovarian tissue by using PBS buffer solution;
s3, placing the cleaned ovarian tissue in a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh tissue of the ovary is 20:1, a step of;
the method comprises the following steps: firstly, filling B1 liquid into a fixing chamber in which the cleaned ovarian tissue is placed for fixing;
after 3 hours of fixing treatment, injecting new B2 liquid for replacing the liquid after partial use, wherein the liquid after partial use is replaced by injecting new B2 liquid once every 0.5 hour of fixing treatment, and the total replacement is 5 times;
after the 5 th time of replacement of the partially used liquid with the new B2 liquid, fixing treatment is performed for 0.5 hour, the new B3 liquid is injected to replace the partially used liquid, and every fixing treatment is performed for 1 hour, the new B3 liquid is injected to replace the partially used liquid for 5 times.
The total fixation time of the first fixation and the second fixation was 13.5 hours.
In examples 2-4, each time a change of the liquid after partial use was performed, the volume of the liquid to be changed was 20% of the total volume of the liquid in the stationary chamber.
Comparative example 1
Fixing fresh tissue of the mammary gland in 95% ethanol water solution (namely, 95% ethanol in the ethanol water solution) with the volume ratio of 95% ethanol water solution to fresh tissue of the mammary gland of 20:1, the fixation time of the mammary tissue was 13.5 hours as the total fixation time of the first fixation and the second fixation in example 2.
Comparative example 2
Fixing the fresh tissue of the thyroid gland in an ethanol water solution with the mass percentage of 95 percent (namely, the mass percentage of ethanol in the ethanol water solution is 95 percent), wherein the volume ratio of the ethanol water solution with the mass percentage of 95 percent to the fresh tissue of the mammary gland is 20: the time for fixation of thyroid tissue was the same as the total fixation time for the first fixation and the second fixation in example 3, i.e., 16 hours.
Comparative example 3
Fixing the fresh tissue of the ovary in 95% ethanol water solution (namely, the ethanol in the ethanol water solution is 95%) by mass percent, wherein the volume ratio of the 95% ethanol water solution to the fresh tissue of the breast is 20: the time for fixation of ovarian tissue was 13.5 hours as the total fixation time for the first fixation and the second fixation in example 4.
Comparative example 4
The method comprises the steps of (1) placing fresh tissues of mammary glands into 10 parts of pentanediol solution with the mass percent of 2.5% (prepared by mixing 10 parts of glutaraldehyde in a glutaraldehyde water solution with 90 parts of phosphate buffer solution) for fixation, wherein the volume ratio of the pentanediol solution with the mass percent of 2.5% to the fresh tissues of the mammary glands is 20: the fixed time was 13.5 hours as the total fixed time of the first and second fixes in example 2.
Example 5
This embodiment discloses a tissue fixation device that may be used in the first fixation procedure of embodiment 4 to replace a partially used fluid.
When the liquid B is used for fixing tissues, the mass percentage of ethanol contained in the liquid in the fixing chamber is gradually increased by replacing part of the used liquid in the fixing chamber, so that gradient fixing and dehydration of the ethanol to the tissues are realized, and the fixing and dehydration effects are good.
Referring to fig. 3, a liquid changing device for fixing fresh tissue includes a liquid tank 1, the liquid tank 1 includes a fixing chamber 11 and a linkage chamber 12, and the liquid used in the fixing chamber 11 is discharged and updated by injecting new liquid into the linkage chamber 12; a movable piston 2 is arranged between the fixed chamber 11 and the linkage chamber 12, a cavity 21 is arranged in the movable piston 2, an opening 22 is formed in the lower surface of the movable piston 2, a movable frame 23 is arranged at the position of the opening 22 of the cavity 21, and the movable frame 23 can seal the opening 22; when new liquid is injected into the linkage chamber 12, the movable piston 2 moves towards the fixed chamber 11, and the same amount of used liquid is discharged from the fixed chamber 11; after a certain amount of new liquid is injected into the linkage chamber 12, the fixed chamber 11 stops draining, the movable frame 23 in the cavity 21 moves, the opening 22 is opened, a hollow chamber 24 is formed, and the hollow chamber 24 is arranged penetrating through the movable piston 2; the bottom of the movable piston 2 is provided with a reset component 3, and the reset component 3 is used for resetting the movable piston 2; the liquid discharging hole 4 is formed in one side of the bottom of the fixed chamber 11, the liquid discharging hole 4 comprises a linkage assembly 5, the linkage assembly 5 is matched with the movable piston 2 to be used for opening and closing the liquid discharging hole 4, and a pressure release wall 13 is arranged on the side wall of the bottom of the fixed chamber 11.
Referring to fig. 4, further, a water inlet pipe 121 is disposed on one side above the linkage chamber 12, a control valve 122 is disposed between the water inlet pipe 121 and the linkage chamber 12, a baffle 123 is disposed on the top of the inner cavity of the linkage chamber 12, so that a liquid injection space is left on the top of the linkage chamber 12, the water inlet pipe 121 is connected with an external fixed liquid injection device, new liquid is injected into the linkage chamber 12, wherein the control valve 122 is set to cooperate with injection equipment, when new liquid needs to be injected into the linkage chamber 12, the control valve 122 is opened, the external new liquid is injected into the linkage chamber 12 through the water inlet pipe 121 and the control valve 122, after the injection is completed, the injection equipment stops working, the control valve 122 is closed at the same time, and the linkage chamber 12 and the outside are cut off, so that the linkage chamber 12 is in a closed state;
a communication valve is arranged on one side above the linkage chamber 12, so that new liquid can be conveniently injected into the linkage chamber 12 for the first time; the front side of the fixing chamber 11 should be provided with a placement opening for conveniently placing fresh tissues, and the placement opening is provided with a sealing component, and the part is the prior art and is not described herein;
in the actual operation process, when liquid is injected into the linkage chamber 12 for the first time, a certain space is reserved above the movable piston 2 due to the baffle 123 arranged at the top of the linkage chamber 12, and then after the liquid tank 1 is filled with new liquid, the space at the baffle 123 is filled.
Referring to fig. 4-6, further, the opening 22 is located in the middle above the cavity 21, the moving frames 23 are respectively located at two sides of the opening 22, an electric push rod 231 is arranged at the outer side of each moving frame 23, the electric push rod 231 is fixed in the cavity 21, an output shaft of the electric push rod 231 is connected with the moving frames 23, support rods 25 are arranged at two sides of the electric push rod 231, the support rods 25 are used for supporting the cavity 21, the moving frames 23 are driven to move through the electric push rod 231, and then the moving frames 23 at two sides are combined and separated, and in the moving process, the cavity 21 is supported through the support rods 25, so that the inside of the cavity 21 is more stable.
Further, the electric push rod 231 drives the moving frames 23 to move, and when the two moving frames 23 are combined, the opening 22 is closed; when the two moving frames 23 are separated, the space of the formed empty chamber 24 is enlarged, so that new liquid in the linkage chamber 12 flows into the empty chamber 24, the pressure of the liquid above the fixed chamber 11 to the moving piston 2 is reduced, the empty chamber 24 is formed at the opening 22 by separating the two moving frames 23, the space of the moving piston 2 is enlarged, the empty chamber 24 is replenished by the liquid in the linkage chamber 12 due to the pressure, the pressure of the liquid in the linkage chamber 12 to the moving piston 2 is reduced, the balance state of the moving piston 2 is broken, at the moment, under the action of the reset assembly 3, the moving piston 2 continuously moves upwards, the space in the linkage chamber 12 is compressed, and the liquid in the linkage chamber 12 flows into the fixed chamber 11 through the empty chamber 24, so that the replenishment of the liquid in the fixed chamber 11 is completed.
Further, reset subassembly 3 includes spacing section of thick bamboo 31, spacing section of thick bamboo 31 is inside to be provided with compression spring 32, when moving piston 2 is located linkage room 12 top, compression spring 32 is in half compression state, compression spring 32 both ends are connected with spacing section of thick bamboo 31 inner wall and fixed room 11 inner chamber bottom respectively, fixed room 11 bottom is provided with guide bar 111, guide bar 111 is located compression spring 32 inside, when moving piston 2 is located linkage room 12 top through being in half compression state's compression spring 32, moving piston 2 receives compression spring 32 ascending elasticity all the time, after moving piston 2 moves a certain distance downwards, further compress compression spring 32, make the elastic potential energy that compression spring 32 possessed further increase, when linkage room 12 and fixed room 11 intercommunication and the pressure difference between them form, compression spring 32 promotes moving piston 2 upwards, the new liquid in the linkage room 12 pours into in the fixed room 11 through empty room 24, wherein when moving piston 2 moves on the line, fixed room 11 space expansion, linkage room 12 space compression, realize pouring the new liquid in the linkage room 12 into the fixed room 11.
Referring to fig. 7-8, further, the linkage assembly 5 includes a linkage bar-shaped tooth 51 and a linkage gear set 52, the linkage bar-shaped tooth 51 is used for driving the linkage gear set 52, the linkage bar-shaped tooth 51 is rotatably connected to one side of the bottom of the moving piston 2, a wedge-shaped bump 511 is arranged at a position below the vertical state of the fixed chamber 11, the inclined plane of the wedge-shaped bump 511 is located at one side of the tooth of the linkage bar-shaped tooth 51, the linkage bar-shaped tooth 51 moves downwards under the driving of the moving piston 2 through the linkage bar-shaped tooth 51, when the bottom of the linkage bar-shaped tooth 51 moves to the wedge-shaped bump 511, the linkage bar-shaped tooth 51 is deflected outwards under the action of the wedge-shaped bump 511, and then the tooth part of the linkage bar-shaped tooth 51 is far away from the linkage gear set 52.
Referring to fig. 9-10, further, the linkage gear set 52 includes a transmission ratchet 521, the transmission ratchet 521 is located below one side of the linkage bar-shaped tooth 51, the transmission ratchet 521 is meshed with the linkage bar-shaped tooth 51, a transmission gear 527 is fixedly arranged on one side of the transmission ratchet 521 coaxially, a driving gear 522 is meshed with the other side below the transmission gear 527, a sector sealing block 523 is arranged on the back of the driving gear 522, the transmission ratchet 521 and the driving gear 522 are both rotatably connected with the inner wall of the fixed chamber 11, and the transmission ratchet 521 is meshed with the linkage bar-shaped tooth 51 through the transmission ratchet 521, so that when the linkage bar-shaped tooth 51 moves downwards, the transmission ratchet 521 is driven to rotate, the transmission gear 527 on the other side of the transmission ratchet 521 is driven to rotate in the same direction, and the driving gear 522 meshed with the transmission gear 527 is driven to rotate, and when the driving gear 522 rotates, the sector sealing block 523 is driven to release the liquid discharging hole 4;
the teeth on the linkage bar teeth 51 are right-angle teeth, the right angle is positioned at the lower side, the ratchet teeth on the transmission ratchet 521 are right-angle teeth, and the right angle is positioned at the upper side, when the linkage bar teeth 51 move downwards, the ratchet teeth on the linkage bar teeth 51 are mutually limited with the ratchet teeth on the transmission ratchet 521, so that the linkage bar teeth 51 are always meshed with the transmission ratchet 521; when the linked bar teeth 51 move upward, one face of the tooth slope on the linked bar teeth 51 repels one face of the tooth slope on the transmission ratchet 521, so that the linked bar teeth 51 deflect outward when the linked bar teeth 51 move upward, and the linked bar teeth 51 cannot transmit with the transmission ratchet 521.
Further, when the linked bar teeth 51 move downward, the frictional force between the ratchet teeth on the linked bar teeth 51 and the ratchet teeth on the driving ratchet 521 is greater than the lateral thrust given by the driving ratchet 521 to the linked bar teeth 51, and the linked bar teeth 51 do not deflect outward.
Further, a second torsion spring is arranged between the linkage bar-shaped tooth 51 and the movable piston 2, and when the linkage bar-shaped tooth 51 moves downwards, the deflection of the linkage bar-shaped tooth 51 is prevented by the elastic limit of the second torsion spring; in the upward reset process of the linked bar teeth 51, reset of the linked bar teeth 51 in the deflected state can be realized by reset of the second torsion spring.
Referring to fig. 9, further, a fan-shaped sealing block 523 is used for sealing the liquid drain hole 4, a fan-shaped through hole 524 is provided on one side of the fan-shaped sealing block 523, the fan-shaped through hole 524 is used for communicating the liquid drain hole 4 with the fixed chamber 11, a fixed rod 41 is provided on one side of the liquid drain hole 4, the fixed rod 41 is located inside the fan-shaped through hole 524, the fixed rod 41 is matched with the fan-shaped through hole 524 to limit the rotation range of the driving gear 522, and the fan-shaped through hole 524 on the other side is matched with the fan-shaped sealing block 523, so that the sealing and the communicating between the liquid drain hole 4 and the fixed chamber 11 are realized under the driving of the driving gear 522.
Further, a torsion spring 525 is further arranged at the back of the driving gear 522, a fixing ring 526 is arranged outside the fan-shaped sealing block 523 and the fan-shaped through hole 524, the fixing ring 526 is fixed on the side wall of the fixing chamber 11, two ends of the torsion spring 525 are respectively connected with the driving gear 522 and the fixing ring 526, when the driving gear 522 is driven to rotate by the transmission gear 527 through the arranged torsion spring 525, the torsion spring 525 deforms and condenses elastic potential energy, and when the driving gear 522 loses the power of the transmission gear 527, the torsion spring 525 releases the elastic potential energy, so that the driving gear 522 is reversely rotated and recovered;
under the action of the fixing rod 41 disposed in the fan-shaped through hole 524, the maximum rotation degree between the fan-shaped sealing block 523 and the fan-shaped through hole 524 only enables the upper and lower positions of the fan-shaped sealing block 523 and the fan-shaped through hole 524 to be interchanged, and the driving gear 522 is reversely rotated and restored under the action of the torsion spring 525, that is, when the liquid discharging hole 4 is blocked by the fan-shaped sealing block 523, the driving gear 522 cannot continuously reversely rotate, and when the linkage bar-shaped tooth 51 moves upwards, the driving gear 522 cannot continuously reversely rotate due to the existence of the fixing rod 41, so that the linkage bar-shaped tooth 51 cannot be transmitted with the transmission ratchet 521, and the linkage bar-shaped tooth 51 moves upwards and simultaneously outwards under the action of the transmission ratchet 521, so that the independent reset of the linkage bar-shaped tooth 51 is realized.
In use, fresh tissue to be fixedly stored is first placed into the interior of the fixing chamber 11 through the placement opening on the front side of the fixing chamber 11.
First liquid injection: the two movable frames 23 are separated through the electric push rod 231, so that an opening 22 above the movable piston 2 is in an open state, then whether the movable piston 2 is at the uppermost part of the inner cavity of the liquid tank 1 is checked, and when the movable piston 2 is at the uppermost part of the inner cavity of the liquid tank 1, the fan-shaped sealing block 523 seals the liquid discharge hole 4, so that the liquid discharge hole 4 is in a closed state; further opening a control valve 122 at one side above the linkage chamber 12 to control the injection of external new liquid into the linkage chamber 12, opening a communication valve at the other side above the linkage chamber 12 to enable the new liquid to flow into the fixed chamber 11 through the empty chamber 24 until the liquid is full of the liquid tank 1, wherein the empty chamber 24 is formed, air in the liquid tank 1 is discharged through the communication valve, when a small amount of liquid overflows from the communication valve, closing the control valve 122 at the water inlet pipe 121, and at the moment, driving the two movable frames 23 to be combined again through the electric push rod 231 to enable the opening 22 above the movable piston 2 to be closed, namely, the empty chamber 24 is closed to enable the liquid in the empty chamber 24 to be discharged and collected, and then closing the communication valve to finish the first injection;
in the actual process, when liquid is injected into the linkage chamber 12 for the first time, a certain space is reserved above the movable piston 2 due to the baffle 123 arranged at the top of the linkage chamber 12, and then after the liquid tank 1 is filled with new liquid, the space at the baffle 123 is filled up;
liquid replacement: firstly, a control valve 122 at a water inlet pipe 121 is opened, new liquid with higher pH value is injected into a linkage chamber 12, a certain space is reserved above a movable piston 2 due to a baffle 123 arranged at the top of the linkage chamber 12, when the new liquid is injected into the linkage chamber 12, the liquid in the space at the baffle 123 is further in a closed state at the moment through an opening 22 above the movable piston 2, when the new liquid is injected into the linkage chamber 12, the movable piston 2 is subjected to pressure and conducts the pressure to the liquid in the fixed chamber 11, after the liquid in the fixed chamber 11 is subjected to pressure, a pressure release wall 13 at one side of the fixed chamber 11 is outwards expanded, the movable piston 2 is downwards moved, the movable piston 2 drives the movable bar tooth 51 to downwards move, and when the movable bar tooth 51 downwards moves, the movable bar tooth 51 is mutually limited by a ratchet tooth on the movable bar tooth 521, the movable bar tooth 521 is always meshed with a driving ratchet tooth 521, when the movable bar tooth 51 downwards moves downwards, the movable bar tooth 521 drives the movable bar tooth 521 to rotate, a sector gear 527 drives a sealing torsion spring 522, and a sealing jaw 522 is further rotatably arranged at the other side of the movable bar tooth 521, and a sealing jaw 522 is rotatably deformed, and a sealing jaw 522 is driven by a sealing jaw 522; the used liquid in the fixed chamber 11 is discharged from the liquid discharge hole 4, when the movable piston 2 moves downwards to the limit cylinder 31 to be propped against the bottom surface of the inner cavity of the fixed chamber 11, the compression spring 32 is compressed into the limit cylinder 31, at the moment, the bottom of the linkage bar-shaped tooth 51 moves to the wedge-shaped convex block 511, the linkage bar-shaped tooth 51 deflects outwards due to the action of the wedge-shaped convex block 511, and then the tooth part of the linkage bar-shaped tooth 51 is far away from the linkage gear set 52, so that the driving gear 522 in the linkage gear set 52 is out of limit, when the driving gear 522 loses the power of the driving gear 527, the torsion spring 525 releases elastic potential energy, so that the driving gear 522 is reversely rotated and recovered, at the moment, the driving gear 522 is reversely rotated, the upper and lower positions of the sector-shaped sealing block 523 and the sector-shaped through hole 524 are exchanged, and the sector-shaped sealing block 523 plugs the liquid discharge hole 4, and at the moment, the control valve 122 is closed;
then, the electric push rod 231 drives the movable frame 23 to move, so that the movable frames 23 on two sides are combined and separated, a cavity 24 is formed at the opening 22, the space of the movable piston 2 is enlarged, the cavity 24 is replenished by the liquid in the linkage chamber 12 due to pressure, the pressure of the liquid in the linkage chamber 12 to the movable piston 2 is reduced, the balance state of the movable piston 2 is broken, at the moment, the movable piston 2 moves upwards all the time under the action of the reset assembly 3, the space in the linkage chamber 12 is compressed, the liquid in the linkage chamber 12 flows into the fixed chamber 11 through the cavity 24, the replenishment of the liquid in the fixed chamber 11 is completed, after the movable piston 2 is pushed to the top of the linkage chamber 12, the electric push rod 231 drives the movable frame 23 to move, the cavity 24 is closed, and the liquid in the cavity 24 is extruded to the top of the linkage chamber 12;
when the movable piston 2 is positioned at the top of the linkage chamber 12 through the compression spring 32 in a semi-compression state, the movable piston 2 is always subjected to upward elasticity of the compression spring 32, when the movable piston 2 moves downwards for a certain distance, the compression spring 32 is further compressed, so that elastic potential energy possessed by the compression spring 32 is further increased, when the linkage chamber 12 is communicated with the fixed chamber 11 and gravity is applied to the upper part of the movable piston 2 to be reduced, the compression spring 32 pushes the movable piston 2 to move upwards, new liquid in the linkage chamber 12 is injected into the fixed chamber 11 through the empty chamber 24, and when the movable piston 2 moves on line, the space of the linkage chamber 12 is compressed, and the space of the fixed chamber 11 is expanded, so that the new liquid in the linkage chamber 12 is injected into the fixed chamber 11;
when the movable piston 2 moves upwards, the linkage bar teeth 51 also move upwards, as the teeth on the linkage bar teeth 51 are right-angle teeth, the right angles are positioned on the upper side, the ratchet teeth on the transmission ratchet 521 are right-angle teeth, and the right angles are positioned on the lower side, when the linkage bar teeth 51 move upwards, one inclined tooth surface on the linkage bar teeth 51 and one inclined tooth surface on the transmission ratchet 521 repel each other, so that the linkage bar teeth 51 deflect outwards when moving upwards, the linkage bar teeth 51 cannot be transmitted with the transmission ratchet 521, and the influence on the tightness of the liquid discharge hole 4 caused when the linkage bar teeth 51 move upwards is avoided.
Further, the difference from the above is that a driving tooth is provided on the linkage bar tooth 51, a linkage tooth is separately provided on one side of the transmission ratchet 521, a fan-shaped tooth is provided on the other side of the transmission ratchet 521, the driving tooth on the linkage bar tooth 51 is matched with the linkage tooth on the transmission ratchet 521, a driving gear 522 is provided on the other side of the transmission ratchet 521, the fan-shaped tooth on the transmission ratchet 521 is meshed with the driving gear 522, and when the linkage bar tooth 51 drives the transmission ratchet 521 to rotate once, the driving gear 522 drives the fan-shaped sealing block 523 to complete the operation of opening the liquid drain hole 4 once.
When the linkage bar-shaped tooth 51 moves downwards until the driving tooth and the linkage tooth are completely misplaced, the transmission ratchet 521 is reset to the drain hole 4 to be closed under the reset action of the torsion spring 525. When the linked bar-shaped tooth 51 is reset upwards, the driving gear 522 is blocked from reversing due to the fixing rod 41, so that the driving gear 522 cannot continuously reverse, transmission cannot be performed between the linked bar-shaped tooth 51 and the transmission ratchet 521, and the linked bar-shaped tooth 51 is rotated outwards while being moved upwards by the acting force of the transmission ratchet 521, and further the independent reset of the linked bar-shaped tooth 51 is realized.
Further, when the interlocking bar-shaped tooth 51 moves downward, the interlocking bar-shaped tooth 51 rotates to be separated from the transmission ratchet 521 under the action of the wedge-shaped lug 511 when the interlocking bar-shaped tooth 51 is matched with the wedge-shaped lug 511, and the driving gear 522 completes rotary reset under the action of the torsion spring 525.
Preferably, a pH detector 14 is also provided on the side of the holding chamber 11 in the device, and a controller is provided for the device to determine the pH of the liquid in the holding chamber 11.
Test examples
The morphology of the treated mammary tissue after the fixation treatment with the fixation liquid prepared in example 1 in example 2, the treated thyroid tissue after the fixation treatment with the fixation liquid prepared in example 1 in example 3, the treated ovarian tissue after the fixation treatment with the fixation liquid prepared in example 1 in example 4, the treated mammary tissue after the fixation treatment with a 95% aqueous ethanol solution (i.e., 95% by mass of ethanol in aqueous ethanol solution) in comparative example 1, the treated thyroid tissue after the fixation treatment with a 95% aqueous ethanol solution (i.e., 95% by mass of ethanol in aqueous ethanol solution) in comparative example 2, the treated ovarian tissue after the fixation treatment with a 95% aqueous ethanol solution (i.e., 95% by mass of ethanol in aqueous ethanol solution) in comparative example 3, the treated mammary tissue after the fixation treatment with a 2.5% glutaraldehyde solution in comparative example 4, and the like were observed by dehydration, transparency, embedding, slicing, and the morphology of the treated tissues was recorded as shown in table 1.
TABLE 1
As can be seen from the analysis of Table 1, the formaldehyde-free fixing solution can be used for fixing a plurality of fresh tissues, has the advantages of no obvious shrinkage of the tissues fixed by the fixing solution, moderate hardness, complete slicing, no cracks, clear structure and distinct layers, is beneficial to pathological observation of the tissues, realizes uniform dehydration of the tissues while fixing, reduces the subsequent dehydration time, and improves the efficiency of pathological examination.
In comparative examples 1 to 3, the fixation was performed with a 95% aqueous ethanol solution (i.e., the mass percentage of ethanol in the aqueous ethanol solution was 95%), and since ethanol was strongly dehydrated, the tissue after fixation was contracted and hardened, affecting the quality of the cut pieces, and not favorable for pathological examination of the tissue, and since high concentration ethanol was used for fixation treatment, the tissue surface was rapidly dehydrated to form a structure similar to a protective film, affecting dehydration of the internal material, and further, when the dehydration treatment was performed, it was not possible to completely dehydrate the tissue, affecting the subsequent transparent and embedding processes, and further affecting the result of the examination of the cut pieces of the tissue.
In comparative example 4, the fresh tissue of the breast is fixed by using a pentanediol solution with a mass percentage of 2.5%, and the pentanediol permeability is poor, so that the tissue fixing speed is low, substances in the tissue are difficult to fix completely, the structural definition and the hierarchical definition of the tissue are poor, in addition, in comparative example 2, the dehydration effect of ethanol is not generated, the complete dehydration process is required after the fixation treatment, and the consumed time is long.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for fixing fresh tissues by using a fixing solution, which is characterized by comprising a solution A and a solution B;
the solution A comprises picric acid and glacial acetic acid;
the liquid B is prepared from the following raw materials: glutaraldehyde, ethanol;
the liquid B comprises liquid B1, liquid B2 and liquid B3;
the B1 liquid is prepared from the following raw materials in parts by weight: 10 parts of glutaraldehyde, 50 parts of ethanol and 40 parts of phosphate buffer;
the B2 liquid is prepared from the following raw materials in parts by weight: 10 parts of glutaraldehyde, 70 parts of ethanol and 20 parts of phosphate buffer solution;
the B3 liquid is prepared from the following raw materials in parts by weight: glutaraldehyde 5 parts, ethanol 95 parts;
the method comprises the following steps:
s1, placing fresh tissues in the solution A, and performing first fixation;
s2, after the first fixation is completed, washing the tissue by using PBS buffer solution;
s3, placing the cleaned tissue in a fixing chamber, adding the liquid B, and performing second fixing;
the step of adding the liquid B comprises the replacement of the liquid after partial use;
a linkage chamber is arranged above the fixed chamber, a movable piston is arranged between the linkage chamber and the fixed chamber, and can move up and down between the linkage chamber and the fixed chamber in a sealing mode when the movable piston is closed, and the movable piston is moved up and down to change the volumes of the fixed chamber and the linkage chamber;
the replacement of the partially used liquid comprises the following steps:
when it is desired to replace a portion of the liquid in the stationary chamber,
s101, filling new liquid into the linkage chamber, moving the piston to descend in a force accumulation mode, driving the linkage assembly to move to the opening of the liquid discharge hole, and discharging part of used liquid in the fixed chamber from the liquid discharge hole in the opening state;
s102, after the movable piston descends to a set position, driving the linkage assembly to move to the liquid discharge hole for reclosing;
s103, opening the movable piston to expose an empty chamber of the movable piston, communicating a fixed chamber, a linkage chamber and the empty chamber, filling new liquid into the empty chamber, releasing the accumulated force, keeping a liquid discharge hole closed while resetting the movable piston until the movable piston is reset, closing the movable piston, and sealing and separating the linkage chamber and the fixed chamber again to finish the replacement of part of used liquid;
the linkage assembly comprises linkage bar-shaped teeth and transmission ratchets; the upper end of the linkage bar-shaped tooth is in running fit with the movable piston; the linkage bar-shaped teeth are in unidirectional linkage with the transmission ratchet teeth;
the up-and-down reciprocating motion of the linked bar-shaped teeth comprises the following steps:
step one, when the linked bar-shaped teeth move downwards, the linked bar-shaped teeth contact with the transmission ratchet teeth to drive the transmission ratchet teeth to rotate in a force storage mode so as to open the liquid discharge hole;
step two, when the linked bar-shaped teeth continue to move downwards to be separated from the transmission ratchet, the transmission ratchet releases the power to reset and rotate, and the liquid discharge hole is closed;
and thirdly, in the upward resetting process of the linked bar-shaped teeth, the transmission ratchet is blocked by one way to limit the reverse rotation of the transmission ratchet, and the linked bar-shaped teeth overcome the blocking of the transmission ratchet in a deflection mode to reset upward.
2. The method for fixing fresh tissues by using a fixing solution according to claim 1, wherein the picric acid is a picric acid saturated solution, and the mass percentage of the picric acid saturated solution to glacial acetic acid is 15:1.
3. the method of fixing fresh tissue according to claim 1, wherein,
and (3) adding the sequence of each solution in the solution B in the step (S3): firstly adding the B1 solution, then adding the B2 solution and finally adding the B3 solution.
4. The method for fixing fresh tissue according to claim 3, wherein the second fixing in S3 includes the following three liquid changing modes sequentially performed:
in the first liquid exchange mode, when the B2 liquid is required to be added into the fixed chamber, if the liquid in the current fixed chamber is the used B1 liquid, discharging part of the liquid in the current fixed chamber and adding the B2 liquid, and performing the first liquid exchange;
in the second liquid changing mode, when the B2 liquid is required to be added into the fixed chamber, if the liquid in the current fixed chamber comprises the used B2 liquid, discharging part of the liquid in the current fixed chamber, adding the B2 liquid, and performing the second liquid changing;
and in the third liquid exchange mode, when the B3 liquid is required to be added into the fixed chamber, discharging part of the liquid in the current fixed chamber, adding the B3 liquid, and carrying out the third liquid exchange.
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