JP2008076347A - Fixing/supporting agent for biopsy specimen, manufacturing method of fixing/supporting agent for biopsy specimen, and fixing/supporting method for biopsy specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fixing/supporting agent for a biopsy specimen, which can gelate stably, and to provide a manufacturing method of the fixing/supporting agent for the biopsy specimen and a fixing/supporting method for the biopsy specimen. <P>SOLUTION: The fixing/supporting agent for the biopsy specimen comprises: alkyl oxidized glucomannan and a preservative. The biopsy specimen is supported by using such the fixing/supporting agent, whereby a stable gelation can be carried out when a gelatinizing agent is added to the biopsy specimen and the fixing/supporting agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biopsy sample fixing support for fixing a biopsy sample so as not to deteriorate, a method for producing the fixing support, and a biopsy sample fixing support method.

In the field of pathology and clinical examination, an embedding support using a high molecular polysaccharide such as mannan has been reported (see, for example, Patent Document 1). In this embedding support using mannan, a biopsy sample is stored in an embedding cassette, a fixing support composed of mannan and an antiseptic is injected into the space around the biopsy sample, and then this is mixed with methanol. By immersing in a gelling agent made of formalin, the fixing support is gelled to integrally support the biopsy sample and the embedding cassette.
Japanese Patent No. 3049537 (paragraphs [0019] to [0027])

  However, in the above-mentioned fixing support using mannan, when the fixing support is gelled, it cannot be stably gelled, and there are uneven portions in the fixed cells. There is a problem that the test sample is dispersed inside the fixing support or the sample is destroyed. In addition, there is a problem that a partial difference occurs in reactivity when the fixing support is gelled.

  The present invention has been made in view of the above-described problems, and is a biopsy sample fixing support capable of stably gelling a fixing support, a method for producing a biopsy sample fixing support, and a biopsy sample. It is an object of the present invention to provide a fixing support method for a test sample.

  In order to achieve the above object, a fixing support for a biopsy sample according to a main aspect of the present invention is characterized by having an alkylated polymer polysaccharide and a preservative.

  By supporting the biopsy sample using the fixing support of such a configuration of the present invention, stable gelation can be performed when a gelling agent is added to the biopsy sample and the fixing support. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support member and the non-uniform reaction during gelation. Accordingly, the biopsy sample can be stably fixed so as not to degenerate.

  A biopsy sample fixing support according to another aspect of the present invention is characterized by having an alkyloxy soy glucomannan and a preservative.

  In the present invention, glucomannan is used as the high molecular polysaccharide as described above, and an alkyloxylated glucomannan obtained by alkylating this can be used as a fixing support. By supporting the biopsy sample using the fixing support of such a configuration of the present invention, stable gelation can be performed when a gelling agent is added to the biopsy sample and the fixing support. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support member and the non-uniform reaction during gelation. Accordingly, the biopsy sample can be stably fixed so as not to degenerate.

  According to one aspect of the present invention, the alkyl is any one of methyl, ethyl, and propyl. Thus, the alkyl may be any one of methyl, ethyl, and propyl.

  According to one aspect of the invention, the preservative comprises formaldehyde. Thus, what has formaldehyde as a preservative can be used.

  The method for producing a fixing support according to the main aspect of the present invention includes a step of oxidizing glucomannan to generate oxidized glucomannan, a step of emulsifying the oxidized glucomannan to generate a first emulsion, and the first emulsification. A step of dehydrating a product, and a step of alkylating the product after the dehydration step.

  The fixing support produced by such a construction of the present invention has an alkylated alkyl oxidised glucomannan. By supporting a biopsy sample using such a fixing support, stable gelation can be performed when a gelling agent is added to the biopsy sample and the fixing support. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support member and the non-uniform reaction during gelation. Accordingly, the biopsy sample can be stably fixed so as not to degenerate.

  According to an aspect of the present invention, the alkylation step is any one of methylation, ethylation, or propylation. Thus, the alkylation step may be any one of methylation, ethylation or propylation.

  According to one aspect of the present invention, after the alkylation step, the product is purified, and this is emulsified with alcohol to form a second emulsion, and the second emulsion is dried and powdered. And a step of dissolving the powder in a preservative. By supporting the biopsy sample using the fixing support thus produced, stable gelation can be performed when a gelling agent is added to the biopsy sample and the fixing support. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support member and the non-uniform reaction during gelation. Accordingly, the biopsy sample can be stably fixed so as not to degenerate.

  According to one aspect of the present invention, in the alkylation step, hydrochloric acid is added to the product obtained in the dehydration step, the mixture is stirred under heating, and then naturally cooled. Thus, by allowing to cool naturally, a self-reaction called so-called aging can be promoted, and alkylation can be sufficiently performed.

  A biopsy sample fixing and supporting method according to a main aspect of the present invention includes a step of accommodating a biopsy sample in an embedding cassette, and a methyl oxysidoglucomannan and a preservative in a space around the biopsy sample. The method includes a step of injecting a fixing support, and a step of gelling by immersing the biopsy sample and the fixing support in a liquid composed of a gelling agent.

  According to such a configuration of the present invention, since a fixing support having a methyl oxydide glucomannan and a preservative is used, a stable gelled state can be obtained when gelling with a gelling agent. Can do. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support member and the non-uniform reaction during gelation. Accordingly, the biopsy sample can be stably fixed so as not to degenerate.

  (Powder for fixing support for biopsy sample and method for producing fixing support for biopsy sample)

  Hereinafter, a method for producing a biopsy sample fixing support powder and a biopsy sample fixing support agent of the present invention will be described with reference to FIGS. In this embodiment, glucomannan is used as the polymer polysaccharide.

  FIG. 1 is a production process diagram of a fixing support powder and a fixing support. FIG. 2 is a diagram showing changes in viscosity in the glucomannan oxidation step. FIG. 3 is a diagram showing the relationship between the rotational speed and processing time of the homogenizer and the viscosity in the first emulsification step. FIG. 4 is a diagram showing the treatment conditions of the present example and the comparative example in the methylation step.

  In the oxidation step (S1), 200 g of cocoon flour was first dissolved in 3 liters of 5 wt% hydrogen peroxide water and reacted for about 7 days under the treatment temperature conditions of 20 ° C., 30 ° C., 40 ° C., and 50 ° C. The dissolved material shows a solidified state at the initial stage of the reaction, but has fluidity and can be stirred as the oxidation proceeds. As shown in FIG. 2, in any treatment temperature condition, stirring was possible on the third day from the start of the reaction, that is, fluidity was observed, and the viscosity of the dissolved material was almost flat after the seventh day. . The viscosity of the melt was measured at a minimum rotation speed of 15 rpm and a viscosity measurement condition of 25 ° C., neglecting the torque of the stirrer. The end of the oxidation step was confirmed by the degree of fluidity, and it was determined that the oxidation step was completed when the viscosity became 65000 mPa · s or less regardless of the treatment temperature. In addition, the processing temperature in an oxidation process has preferable 20-50 degreeC, More preferably, the processing temperature of 35-45 degreeC is optimal. When the treatment temperature was lower than 20 ° C., the reaction rate was remarkably lowered, and it took 14 days to complete the oxidation reaction. On the contrary, an extremely low viscosity state that was not expected to be higher than 50 ° C. was exhibited. This oxidation step produces oxidized glucomannan.

  Next, in the first emulsification step (S2), an equivalent amount of oxidized glucomannan obtained in the oxidation step is added to 2 liters of methanol having a purity of 99.5% for aggregation. The aggregate is emulsified with a homogenizer. At this time, in order to prevent agglomeration of agglomerates, the homogenizer used for refining was a fine generator-mounted type and emulsified under a processing condition of 5000 rpm for 15 minutes. When the treatment was performed for more than 15 minutes, the first emulsion became gummy due to the frictional heat of the generator, and the reaction in the subsequent steps became impossible. In addition, since the time until it becomes a gum shape varies depending on the homogenizer model and processing conditions, it is necessary to set the processing conditions so as not to change to a gum shape. As shown in FIG. 3, the progress of emulsification is almost completed in 15 minutes at any rotational speed. The completion of the emulsification step was confirmed by liquid viscosity, and it was determined that the first emulsification step was completed when the viscosity became 1000 mPa · s or less at a viscosity measurement temperature of 25 ° C. A 1st emulsion is produced | generated by this 1st emulsification process.

  Next, in the dehydration step (S3), methanol is added to the first emulsion obtained in the first emulsification step, distillation is performed using an evaporator, mixed dehydration is performed five times, and this is immersed in methanol. Glucomannan. The completion of the dehydration step was confirmed by igniting the obtained solution with a pseudo alcohol lamp. If it was not ignited with a pseudo alcohol lamp, it was judged that dehydration was not completely completed. In this embodiment, in the dehydration 1 to 4 times, the ignition by the pseudo alcohol lamp was not confirmed, and the dehydration was not completely completed. If the dehydration is not completely completed, the methylation process may be inhibited and the reaction rate may be reduced in the methylation step.

  Next, in the methylation step (S4), 15N hydrochloric acid is added to methanol-immersed glucomannan, which is a product obtained in the dehydration step, to obtain 0.75 vol. The methylation is carried out by reacting for about 5 hours in a hot water bath at 60 ° C. and stirring at a rotation speed of 200 rpm. Thereafter, the heating was stopped, and the reaction was stopped by allowing the bath to stand in a water bath and lowering the temperature to room temperature. In order to ensure methylation, the concentration after addition of 15N hydrochloric acid is 0.5 to 0.8 vol. %, The reaction temperature is set to 60 to 65 ° C., and the stirring is set to an arbitrary number of revolutions in such a range that the reactant does not overflow from the reactor. Since the reaction promotes self-reaction called so-called aging, the reaction is not forcibly stopped by adding cold water, and the reaction is stopped by cooling the hot water bath overheating the reactor to 15-30 ° C. It is preferable to do. On the other hand, as a comparative example, 15N hydrochloric acid was added to methanol-immersed glucomannan obtained in the dehydration step and 1.0 vol. The methylation was carried out by reacting in a hot water bath at 55 ° C. for about 2 hours without stirring. Thereafter, water was added to lower the temperature and forcibly stop the reaction. In the product of the comparative example thus obtained, the formation of a massive substance in the system was confirmed. When the fixing support produced from this product was observed for 24 hours, yellowing of the system and odor of hydrochloric acid were observed. This is probably because methylation is not completed.

  Next, in the purification step (S5), the methylated product obtained in the methylation step is dispersed in pure water, and is stirred and washed in a vacuum state using an evaporator under heating at 55 ° C. Thereby, hydrochloric acid which is an unreacted oxidant is gasified and removed, and chloride is separated. This was filtered at room temperature, and the resulting solution was filtered again at room temperature to obtain a purified product in order to remove the residue that was deposited after losing hydrochloric acid.

  Next, in the second emulsification step (S6), the purified product obtained in the purification step was emulsified with methanol having a purity of 99.5% or more to obtain a second emulsion.

  Next, in the drying and powdering step (S7), the second emulsion obtained in the second emulsifying step is dried to obtain a powder. As a result, a powder for a fixing support of a biopsy sample made of methyloxyseidoglucomannan is produced.

  Next, in the dissolving step (S8), the fixing support powder is dissolved in an aqueous formaldehyde solution having an arbitrary concentration, for example, 15 to 75%. As a result, a fixing support having methyl oxysidoglucomannan as an alkylated polymer polysaccharide and an aqueous formaldehyde solution as a preservative is obtained.

  (Biopsy sample fixation support method)

  Next, a method for fixing and supporting a biopsy sample using the fixing support manufactured by the above-described manufacturing method will be described with reference to FIGS.

  FIG. 5 is a perspective view of the embedding cassette, and illustration of the lid 3 is omitted. FIG. 6 is a longitudinal sectional view showing a process of injecting the fixing support into the embedding cassette. FIG. 7 is a longitudinal sectional view showing the gelation process. FIG. 8 is a longitudinal sectional view showing a process of dehydrating, degreasing, and penetrating the embedding agent. FIG. 9 is a longitudinal sectional view showing a state of a biopsy sample embedded in a fixing support. FIG. 10 is a longitudinal cross-sectional view showing a process of embedding a biopsy sample. FIG. 11 is a longitudinal sectional view showing the state of the biopsy sample after the embedding process of FIG. 10 is completed. FIG. 12 is a longitudinal sectional view showing a state in which the biopsy sample shown in FIG. 10 is sliced. FIG. 13 is a perspective view showing a sliced slice obtained by FIG.

  First, an embedding cassette for storing a biopsy sample will be described with reference to FIGS. This embedding cassette accommodates a biopsy sample taken from a human body so as not to degenerate.

  The embedding cassette 4 is formed by projecting a lower edge 43 of a flat container 42 toward the lower surface of the planar frame 41 at the center of a rectangular planar frame 41. At least one of the lid 3 and the bottom surface 1 of the container 42 is formed of a transparent porous plate 2. A projecting edge 44 is provided on the upper surface of the planar frame body 41 around the opening edge of the container 42, and communication holes 45 are provided in the planar frame body 41 around the container 42 so as to communicate with both upper and lower surfaces. Yes. A front hooking piece 46 is provided at the front end of the embedding cassette 4 and a rear hooking piece 47 is provided at the rear end, and the lid 3 is detachably fitted thereto. The embedding cassette 4 is made of a synthetic resin.

  Next, a method for supporting fixation of a biopsy sample will be described.

  First, as shown in FIG. 6, a flat embedding cassette 4 with a lid 3 formed of a porous plate 2 having a transparent bottom surface 1 is placed on a petri dish 12. Then, the biopsy sample 5 is accommodated in the embedding cassette 4, and the fixing support 6 manufactured by the above-described manufacturing method is placed from the fixing support container 13 to the arrow A6 above and around the biopsy sample 5. Pour and fill as shown. As a result, the biopsy sample 5 is fixed so as not to be altered and supported by the embedding cassette 4. At this time, the biopsy sample 5 desired by the examiner, for example, a clinician, is supported by the fixing support 6 so that the test surface of the biopsy sample 5 adheres to the bottom surface 1 of the embedding cassette 4.

  Here, the fixation of the fixing support is to fix the raw tissue so that it does not change over time by using the coagulation action of the protein, and to keep the form of each component of the biopsy sample close to that before birth. is there. Further, the term “support” means that the biopsy sample is securely adhered and kept in position so as not to move to the upper surface of the bottom plate in the embedding cassette.

  6 is a gel composed of 99.5% methanol and formalin placed in a capsule 14 and its lid 15 as shown in FIG. The biopsy sample 5 is integrally supported in the embedding cassette 4 by being immersed in the agent 7 and gelling the fixing support 6. In this state, for example, the embedding cassette 4 is transported from a clinician room to a pathology room by mail or the like. Here, the fixing support 6 is gelled by being immersed in the gelling agent 7, but in the present embodiment, since the fixing support 6 having methyl oxysidized glucomannan and an aqueous formaldehyde solution is used, In the gelation with the gelling agent 7, stable gelation can be performed. That is, by methylating glucomannan, the hydrophilicity is lowered and a viscoelastic body is formed without intrusion of surrounding water, and stable gelation can be performed. Thereby, it is possible to suppress or prevent the dispersion or destruction of the biopsy sample inside the fixing support and the non-uniform reaction during gelation.

  As the gelling agent 7, in addition to those described above, alcohols such as alcohol formalin, alcohol, polyethylene glycol, glycerin, and acetone are used.

  In the pathology room, the biopsy sample 5 is taken out from the capsule 14 while being fixed to the embedding cassette 4, and as shown in FIG. 8, high-dehydration treatment of alcohol or the like in the processing container 9 prepared in advance there It is immersed in the agent 8a and dehydrated. After that, it is sequentially immersed in the degreasing and transparent processing agents 8b and 8c in another processing container (not shown). Thereby, dehydration, degreasing, and penetration are performed, and a molten embedding agent made of paraffin or resin is infiltrated.

  Thereafter, the embedding cassette 4 is taken out from the processing container 9, and the biopsy sample 5 infiltrated with the molten embedding agent is embedded in the fixing support 6 from the embedding cassette 4 as shown in FIG. The gel block 36 is taken out and placed on the embedding dish 10 as shown in FIG.

  An empty embedding cassette 4 is placed on an embedding dish 10 at the upper part of the biopsy sample 5, and molten paraffin 11 is injected into them as an embedding agent from above, and cooled and solidified by cooling it. The biopsy sample 5 embedded in the fixing support is embedded in the solidified paraffin 11 together with a part of the embedding cassette 4.

  After the paraffin 11 is hardened, the embedding cassette 4 is removed from the embedding dish 100 in the state shown in FIG. As a result, as shown in FIG. 11, the embedding block 18 of the biopsy sample in which the perforated plate 2 on the bottom surface 1 of the embedding cassette 4 is cast with the paraffin 11 and the biopsy sample 5 is embedded therein. Get.

  Thereafter, the embedding block 18 is held by the support base 19 of FIG. 12, and the microtome blade 17 is reciprocated in the direction of the arrow A17 to make a thin slice 20 of the biopsy sample of FIG.

  As described above, in this embodiment, by using a fixing support having methyl oxydide glucomannan and an aqueous formaldehyde solution as a fixing support, stable gelation can be performed, and a biopsy inside the fixing support is performed. It becomes possible to suppress or prevent the dispersion or destruction of the sample and the non-uniform reaction during gelation.

  In the present embodiment, methyloxy soy glucomannan was used as the fixing support powder, but ethyl oxy soy glucomannan and propyl oxidised glucomannan can be used, and a gelling agent was added in the same manner. Stable gelation can be performed.

  In this embodiment, methyl oxysidoglucomannan is used as the alkylated polymer polysaccharide. However, it is also possible to use an alkylated polymer agar or gelatin as the polymer polysaccharide. .

The manufacturing process figure of the fixing support agent in this embodiment, and the powder for fixing support agents of a biopsy sample. The figure which shows the viscosity change in a glucomannan oxidation process. The figure which shows the relationship between the rotational speed and processing time of a homogenizer in a 1st emulsification process, and a viscosity. The figure which shows the process conditions in the present Example and comparative example in a methylation process. The perspective view of an embedding cassette. The longitudinal cross-sectional view which shows the process of inject | pouring a fixing support agent into an embedding cassette. The longitudinal cross-sectional view which shows a gelatinization process. The longitudinal cross-sectional view which shows the process of dehydrating, degreasing, and seeping through the embedding agent. The longitudinal cross-sectional view which shows the state of the biopsy sample embed | buried under the fixing support agent. The longitudinal section which shows the embedding process of a biopsy sample. The longitudinal cross-sectional view which shows the state of the biopsy sample after finishing the embedding process of FIG. The longitudinal cross-sectional view of the state which slices the biopsy sample shown in FIG. The perspective view which shows the sliced slice piece obtained by FIG.

Explanation of symbols

  6 Fixing support

Claims (9)

An alkylated polymeric polysaccharide;
A biopsy sample fixing support comprising a preservative. Alkyl oxysidized glucomannan,
A biopsy sample fixing support comprising a preservative. 3. The fixing support for a biopsy sample according to claim 1, wherein the alkyl is any one of methyl, ethyl, and propyl. The biopsy sample fixing support according to any one of claims 1 to 3, wherein the preservative contains formaldehyde. Oxidizing glucomannan to produce oxidized glucomannan;
Emulsifying the oxidized glucomannan to produce a first emulsion;
Dehydrating the first emulsion;
And a step of alkylating the product after the dehydration step. The method for producing a fixing support for a biopsy sample according to claim 5, wherein the alkylation step is any one of methylation, ethylation, and propylation. After the alkylation step, purify the product and emulsify it with alcohol to form a second emulsion,
Drying the second emulsion to powder;
The method for producing a fixing support for a biopsy sample according to claim 5 or 6, further comprising the step of dissolving the powder in a preservative. 8. The alkylation step according to claim 5, wherein hydrochloric acid is added to the product obtained in the dehydration step and stirred under heating, and then naturally cooled. 9. A method for producing a fixing support for a biopsy sample. Storing the biopsy sample in an embedding cassette;
Injecting a fixing support having methyl oxysidoglucomannan and preservative into the space around the biopsy sample;
A method of fixing and supporting a biopsy sample, comprising the step of gelling the biopsy sample and the fixing support agent by immersing them in a liquid comprising a gelling agent.

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