JP2008129000A - Method and device for paraffin-masking tissue sliced piece - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem wherein a tissue piece specimen is required to be prepared each time for observing a tissue over a long period, because a tissue sliced piece cut thinly from a paraffin embedded block of an extraphysiologic tissue by a microtome or the like is denatured and deteriorated in its tissue structure by influences of oxidation, humidity or the like allowing no storage over the long period as the specimen when stored for the long period under such a condition as it is, and to provide a method and a device capable of preventing tissue activity from being denatured and deteriorated even when the tissue piece is stored for the long period. <P>SOLUTION: A surface of the specimen bonded on a glass substrate with the tissue sliced piece cut thinly from the paraffin embedded block of the extraphysiologic tissue is coated with a paraffin thin film, using a transfer roll rotated while holding molten paraffin on its surface, to mask the specimen with the paraffin. The specimen usable for histologic function analysis over the long period is obtained thereby even when the tissue piece specimens of large number are prepared in once time from the paraffin block, as an effect by this manner. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method and an apparatus for coating a thin tissue tissue slice (hereinafter referred to as “tissue piece”), which is produced when observing pathological tissue cells or the like of a living body with a microscope or the like, with paraffin. .

  In order to prepare a tissue specimen for microscopic observation of a pathological cell tissue or the like in a living body, first, a specimen sample called a normal paraffin block in which a pathological tissue is paraffin infiltrated and embedded in paraffin according to a predetermined procedure is prepared. A piece of tissue having a thickness of about 3 μm is produced from the block using a thin cutting device such as a microtome. The reason for embedding the tissue in paraffin is that it is easy to slice in order to make a tissue piece specimen having a thickness of about 3 μm that can be observed with a microscope, and to prevent deterioration of cellular tissue over time. However, when a large amount of tissue specimens are prepared from this sample at once, the tissue structure deteriorates over time due to modifications such as oxidation and humidity in the atmosphere, so it cannot be stored for a long time. It took time and effort to slice from. The present invention provides a method and apparatus for producing a tissue piece that can be stored for a long period of time without causing tissue deterioration even if a large number of tissue piece specimens are produced from a paraffin block at the same time.

  The tissue pieces sliced from the paraffin block to a thickness of about 3 μm to 5 μm are usually attached to a glass substrate having a width of about 2.5 cm and a length of about 7.5 cm while floating and floating in water. The glass substrate on which the tissue piece is adhered is hereinafter referred to as “glass specimen”. Usually, tissue pieces sliced from a paraffin block are put together in this glass specimen state and put into a special case and stored in the air or in a low-temperature storage. When actually used in microscopic observation, the paraffin is removed from the glass specimen using a deparaffinizing solvent such as xylene, and the specimen is then subjected to a staining process for facilitating discrimination of the tissue. The tissue piece on the dried glass specimen is infiltrated with paraffin, but the sliced surface is exposed to the atmosphere and is modified by oxidation, humidity, etc., and the tissue deteriorates with time, and correct tissue diagnosis There was a drawback to inhibit.

There has been no practical method or apparatus for storing this glass specimen as it is for a long time without deterioration of the tissue over time.
Very rarely, a method for preventing the deterioration of the glass specimen over time is a method in which the glass specimen is immersed in a molten paraffin bath and embedded in paraffin. However, in this method, unnecessary paraffin adheres to the back and side surfaces of the glass, and since the adhesion thickness cannot be controlled and adheres relatively thickly, a large amount of solvent is required in the subsequent deparaffinization process, and the solvent cost is reduced. In addition, since a large amount of waste solvent is discharged, the environmental load is large and the deparaffinization process takes a long time.

The problem to be solved is to propose a method and apparatus that can preserve a glass specimen for a long period of time with little deterioration and deterioration of tissue cells. Specifically, a tissue piece is covered with an ultrathin paraffin film (hereinafter referred to as “ A method and apparatus for “masking” are provided. When masking a tissue piece on a glass specimen with paraffin, it is practically preferable to selectively mask only a portion of the tissue piece on the glass specimen with a paraffin film thickness of 30 μm or less, preferably 20 μm or less. .
According to the present invention, only a portion of the tissue piece can be masked with a uniform paraffin thin film, so that the amount of paraffin used can be reduced, the amount of solvent required for deparaffinization can be reduced, the environmental load can be reduced, and the time required for the deparaffinization process can be reduced. Can also reduce paraffin masking.

  In the present invention, a paraffin is transferred onto a tissue piece of a glass specimen by rotating a rotating roll (hereinafter referred to as “transfer roll”) holding molten paraffin on the surface to which the tissue piece of the glass specimen is adhered. A method and apparatus for paraffin masking a tissue piece with an extremely thin uniform thickness of 30 μm or less are provided.

  That is, according to the present invention, a glass substrate to which a tissue piece sliced from a paraffin block is attached, that is, a glass specimen, is first inserted and set in a substrate holder having an up-and-down lifting means provided on the transfer roll. The substrate holder is provided with a pair of guide means (hereinafter referred to as guide rails) having an “L” -shaped or “U” -shaped cross-sectional shape that holds and guides both ends of the glass specimen. With the tissue piece attached surface down, the tissue piece is inserted horizontally into the guide rail until the position of the tissue piece is deeper than the top of the transfer roll. The guide rail preferably has a length in the insertion direction for receiving the entire length of the glass specimen, and the inserted glass specimen is slidable along the guide rail. A columnar member or a rotatable roll member may be arranged in a line instead of the guide rail. The transfer roll is provided with driving means for rotating it.

  The lower part of the transfer roll is always immersed in a molten paraffin bath, and the molten paraffin is always attached to the surface of the transfer roll in the form of a thin film by the rotation of the transfer roll and lifted upward. The molten paraffin bath is provided with heating means and temperature control means for melting paraffin.

The substrate holder is provided with a feeding roll at the upper part near the uppermost position of the transfer roll and close to the upper surface of the inserted glass specimen, and is fed when the substrate holder is pushed down by the vertical lifting means. The roll is in contact with the upper surface of the glass specimen, that is, the side to which the tissue piece is not adhered, and the glass specimen is pressed against the transfer roll. At the same time, the transfer roll and the feed roll shaft are clutched so that the feed roll rotates in synchronization with the transfer roll. A pair of meshing gears is provided as means. The pitch circle diameter of the meshing gear is set to be approximately equal to the outer diameter of each roll. The clutch means may be means other than the meshing gear as long as the rotation of the transfer roll can be transmitted to the sending roll. Further, it is preferable that the rotation speed (circumferential speed) of the transfer roll and the rotation speed of the feed roll (peripheral speed, which is equal to the feed speed of the glass specimen described later) are substantially the same. For example, the outer diameter of the transfer roll The glass sample may be sent out at a linear speed different from the rotational speed (peripheral speed) of the transfer roll by changing the outer diameter of the feed roll or changing the gear ratio of the meshing gear to other than 1.
The substrate holder includes a spring mechanism as an up-down mechanism, and the substrate holder is normally held at a position where the glass specimen does not touch the transfer roll by the biasing force of the spring mechanism.

In this state, the glass specimen is inserted and set in the substrate holder, and when the substrate holder is pushed down against the urging force of the spring mechanism by the handle, the glass specimen is brought into close contact with the upper surface of the transfer roll with an appropriate contact pressure, and the transfer roll surface is melted. The paraffin is transferred onto the glass specimen, and the tissue piece can be masked with the paraffin thin film. At the same time, the feeding roll is rotated by the meshing gear, and the glass specimen is horizontally inserted along the guide rail. Sent to the side.
If the glass specimen is sent out to the front in the rotation direction of the transfer roll, the part on the back side from the initially set glass specimen position is masked with paraffin, and the necessary minimum paraffin masking including tissue pieces is possible. Become. The surface material of the feed roll that contacts the glass specimen is preferably a rubber-based material or a soft plastic.

  Of course, the same effect can be obtained by transferring the paraffin while moving the glass specimen directly in contact with the surface of the transfer roll that is directly rotated by a human hand without using the substrate holder. A method using a substrate holder as described above is more desirable in terms of the uniformity, repeatability, and operability of the paraffin transfer film.

  Normally, the width of the glass specimen is slightly smaller than the width of the transfer roll that comes into contact with it, so the guide rail that grips the glass specimen before the substrate holder descends and the glass specimen supported by the guide rail contacts the transfer roll. Since it will interfere with the transfer roll first, it is necessary to cut out a part of the guide rail.

  Although it is desirable that the thickness of the paraffin film for masking the tissue piece is thin from the viewpoint of the cost of the paraffin raw material and the deparaffinization solvent, if the thickness is too thin, the tissue is partially exposed and it is difficult to obtain the preservation effect. In the experiments by the inventors, it is usually desirable that the thickness is about 10 μm to 30 μm.

  There are several ways to adjust the thickness of the paraffin film that masks tissue pieces.

  First is the temperature of the molten paraffin. The higher the melting temperature (Melting Point) of the paraffin used, the thinner the film attached to the transfer roll. In the experiments by the inventors, it is desirable to raise the paraffin bath by about 10 to 40 ° C. above the melting temperature. If it is too high, the tissue piece will be damaged and the viscosity of the melted paraffin will be too small, and the transfer film thickness will tend to vary.

  Next, there is a method of using a jig for adjusting a gap with the transfer roll surface, usually called a doctor knife, which controls the thickness of the paraffin film adhering to the transfer roll surface. By adjusting the gap distance between the transfer roll surface and the doctor knife, the paraffin film thickness attached to the transfer roll can be adjusted, and as a result, the paraffin film thickness transferred to the glass specimen is adjusted.

  The outer diameter and rotation speed of the transfer roll also affect the thickness of the paraffin transferred to the glass specimen. When the outer diameter is large and the rotation speed is slow, it takes a long time for the molten paraffin adhering to the roll surface to come into contact with the glass specimen, and it is easy to drop by gravity on the way, resulting in a thin transfer film thickness. On the other hand, if the time to contact is too long, the molten paraffin on the surface of the transfer roll is likely to be cooled and solidified in the air, and the film tends to be transferred to the glass specimen as it is. An appropriate value of the surface peripheral speed of the transfer roll is determined based on this balance, but in the experiments by the inventors, the surface peripheral speed of the transfer roll is desirably about 50 to 300 cm per minute including operability and productivity.

  In addition, the transfer film thickness can also be changed by changing the ratio between the peripheral surface speed of the transfer roll and the linear feeding speed of the glass specimen to other than 1. This ratio can be easily changed by changing the ratio of the meshing gear. If the linear feeding speed of the glass specimen is made larger than the peripheral speed of the transfer roll surface, the transfer film thickness can be easily reduced. However, it should be noted that if the ratio between the two is increased too much, the transfer film thickness becomes uneven. For example, it can be 5 times or less, preferably 1 to 3 times. However, it may be outside this range depending on the peripheral speed of the transfer roll surface.

  The contact pressure and the temperature of the glass specimen itself also affect the film thickness. The stronger the contact pressure is, the thinner the film thickness is. However, care must be taken because it tends to adversely affect the tissue piece. The higher the temperature of the glass specimen, the thinner the transferred film thickness. However, if the film thickness is too thin, it becomes difficult to mask all the tissue pieces. In addition, the glass sample temperature is usually room temperature, but it is effective to preheat the glass sample when it is desired to make the film thickness extremely thin.

  FIG. 1 shows the relationship between the temperature of the paraffin bath and the transfer film thickness on the glass sample when the melting point of 55 to 57 ° C. paraffin is used, and the transfer roller peripheral speed of 78.5 cm / It is the graph measured by min. In this case, the outer diameter of the transfer roll was 50 mm, the immersion length in the paraffin bath was 1/3 of the entire circumference, and the gap between the roll and the doctor knife was 30 μm. It can be seen that the paraffin transfer film thickness decreases as the paraffin temperature increases. If the paraffin bath temperature is 80 ° C. and the roll peripheral speed is 78.5 cm / min, the paraffin transfer film thickness is about 20 μm, and practical paraffin masking can be obtained.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 2 is a front view of an apparatus showing an embodiment of the present invention. FIG. 3 is a side view of the same.

  A tissue piece 5 is affixed to the lower surface of the glass specimen 1, and the tissue piece 5 itself is in contact with the transfer roll 8 in the guide groove 4 of the guide rail 3 provided on the substrate holder 2 having the vertical lifting mechanism 15. Inserted and set so that it is behind the contact position. The substrate holder 2 is provided with a delivery roll 6. The shafts of the delivery roll 6 and the transfer roll 8 are provided with meshing gears 7 and 9 as clutch means. When the substrate holder 2 is lowered, the meshing gear 7 and 9 is engaged, and the feed roll 6 rotates in the reverse direction to the transfer roll 8. At the same time, the feed roll 6 and the transfer roll 8 hold the glass specimen 1 and serve to feed the glass specimen 1 to the insertion side.

  At this time, if the axis of the feed roll 6 is displaced about 1 to 3 mm from the axis of the transfer roll 8 in the insertion direction, when the glass specimen 1 is sent out, the last part is easily separated from the transfer roll 8, and the last It is possible to prevent so-called bulking phenomenon in which the thickness of the paraffin transfer film at the portion increases.

The lower part of the transfer roll 8 is immersed in a molten paraffin bath 10, and the molten paraffin adheres to the surface of the transfer roll by rotation and is lifted into a thin film, and transferred to the glass specimen 1 at the uppermost part.
11 is a heater, 12 is a heater temperature adjusting device, 13 is a paraffin bath, 14 is a drive device for the transfer roll 8, and 15 is a vertical lift mechanism for the substrate holder 2. A spring mechanism 18 for adjusting a contact pressure when the glass specimen 1 is brought into contact with the transfer roll 8 is incorporated in the up-and-down lift mechanism 15, and the spring mechanism is usually located at a position where the glass specimen 1 does not come into contact with the transfer roll 8. It is held with an urging force of 18. Reference numeral 19 denotes a handle for moving the entire substrate holder 2 up and down manually.

  When the handle 19 is pushed down and the substrate holder 2 is lowered by the up-and-down lifting mechanism 15 to bring the glass specimen 1 into contact with the transfer roll 8, the paraffin film is transferred to the glass specimen 1 and simultaneously the glass specimen 1 is fed out and the transfer roll 6 and transfer roll. The glass sample 1 is horizontally held to the insertion side by the rotation of the two. Reference numeral 16 denotes a doctor knife that adjusts the paraffin film thickness on the transfer roll surface, and 17 denotes a scraper that constantly contacts the transfer roll surface and scrapes off the paraffin that is fixed to the transfer roll.

2 and 3, an aluminum transfer roll having an outer diameter of 50 mm and a surface length of 30 mm is rotated using a glass specimen in which a tissue piece having a thickness of 3 μm is attached to a slide glass having a width of 25 mm, a length of 75 mm, and a thickness of 1 mm. A glass sample is prepared by immersing one third of the entire circumference of the roll in a molten paraffin bath set to 5 revolutions per minute (circumferential speed 78.5 cm / min) and controlling a paraffin having a melting point of 55-57 ° C. to 80 ° C. When the paraffin was transferred, the thickness of the transferred paraffin was within the range of 20 μm ± 3 μm, and the tissue piece could be uniformly masked with paraffin.
The gap between the transfer roll and the doctor knife at that time was adjusted to 15 μm. Further, the outer diameter of the feed roll was 25 mm, the surface length was 25 mm, and the surface material was soft rubber.

  It is a graph which shows a paraffin melting temperature and a paraffin transfer film thickness.   It is a front view of the apparatus which shows this invention.   It is a side view of the apparatus which shows this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Glass specimen 2 Substrate holder 3 Guide rail 4 Guide groove 5 Tissue piece 6 Feeding roll 7 Meshing gear 8 Transfer roll 9 Meshing gear 10 Molten paraffin bath 11 Heating heater 12 Heater temperature control device 13 Paraffin bathtub 14 Drive device 15 Vertical lift mechanism 16 Doctor knife 17 Scraper 18 Spring mechanism 19 Handle

Claims (8)

  A paraffin masking method for a tissue thin section, wherein the substrate is coated with a paraffin film having a thickness of 30 μm or less by rotating a transfer roll holding molten paraffin in close contact with the substrate surface to which the tissue thin section is adhered.   The method according to claim 1, wherein the tissue thin section is cut from a paraffin-embedded body of a pathological tissue.   Hold the substrate with the tissue slice attached so that the tissue piece attachment side is the bottom surface, and move the substrate horizontally along the direction of rotation while rotating the transfer roll holding the molten paraffin in close contact with the bottom surface. The method according to claim 1 or 2, wherein a uniform paraffin film is formed.   A substrate holder including a delivery roll for horizontally moving a substrate on which a tissue slice is adhered, and a vertically movable substrate holder, a transfer roll for holding molten paraffin, a driving means for rotating the substrate holder, and a substrate and a transfer roll in the substrate holder Clutch means for transmitting the driving force of the driving means to the delivery roll when contacted, and when the substrate is inserted into the substrate holder and lowered, the lower surface of the substrate comes into contact with the transfer roll and the molten paraffin is applied to the substrate. A paraffin masking apparatus characterized in that, simultaneously with transfer, a substrate is sandwiched between a delivery roll and a transfer roll, and is horizontally moved by rotation of the delivery roll or the delivery roll and the transfer roll and delivered from the substrate holder.   The substrate holder has guide means for receiving and holding the substrate, and the clutch means is a first meshing gear provided on the rotation shaft of the feed roll and a second meshing gear provided on the rotation shaft of the transfer roll. When the substrate holder is lowered, the upper surface of the transfer roll comes into close contact with the lower surface of the substrate in the guide means, and the first meshing gear and the second meshing gear mesh with each other. 5. The paraffin masking apparatus according to claim 4, wherein the driving force of the driving means is transmitted to the sending roll and rotated to move the substrate in the guiding means in the horizontal direction in accordance with the rotation of the transfer roll.   Further, the up-and-down lifting mechanism includes a spring mechanism that urges the substrate holder upward so that the substrate holder is separated from the transfer roll, and the substrate holder includes a handle for pushing down the substrate holder against the urging force of the spring mechanism. The paraffin masking apparatus according to claim 5. A substrate guide means for holding the tissue-sliced substrate movably in the horizontal direction and a feed roll which is pivotally supported above the substrate guide means and moves the substrate in the guide means in the horizontal direction when rotated. Vertically movable substrate holder, including
A molten paraffin bath with heating means to melt the paraffin,
A transfer roll having at least a part of its surface positioned in the molten paraffin bath and pivotally supported below the substrate guiding means substantially parallel to the delivery roll;
Drive means for rotating the transfer roll;
A vertical lift mechanism for moving the substrate holder up and down between a first position where the substrate does not contact the transfer roll and a second position where the substrate contacts the transfer roll, and a first shaft provided on the rotating shaft of the feed roll And a second meshing gear provided on the rotation shaft of the transfer roll,
When the substrate holder held in the first position by the spring means is pushed down from the first position to the second position by the handle, the upper surface of the transfer roll comes into close contact with the lower surface of the substrate in the guide means. At the same time, when the first meshing gear and the second meshing gear mesh with each other, the driving force of the driving means is transmitted to the sending roll to rotate it, and the substrate in the guiding means is rotated according to the rotation of the transfer roll. The paraffin masking device according to claim 6, which is configured to move in a horizontal direction.   The paraffin masking device according to claim 7, wherein the axis of the feed roll is displaced in the substrate insertion direction from the axis of the transfer roll.

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