Plate guide type arrayer for producing tissue array blocks

Abstract

An object of the present invention is to provide a plate-guided arrayer for producing tissue array blocks. The present invention relates to an arrayer for producing tissue arrays, comprising a recipient block holder with an upper surface and a cavity for receiving a tissue recipient block, and a guide plate designed to be joined to the upper surface of the recipient block holder. The guide plate includes an array of through holes aligned with the cavity of the recipient block holder. The recipient block holder and the guide plate are designed to be fixed to each other in a fixing assembly. The present invention also provides a full set comprising the arrayer and a punch pen for forming holes in the recipient block and transferring tissue to the recipient block holder. The present invention also provides a method of using the arrayer and the set.

Description

Background Art

[0001] A tissue array or tissue microarray (TMA) typically refers to a recipient block in which hundreds of individual cylindrical tissue cores are aligned. Each of these recipient blocks can be cut into hundreds of thin sections (TMA tissue sections), which can then be used for high-throughput immunohistochemistry or in situ hybridization analysis of proteins, RNA, or DNA molecules in cells or tissues. TMA technology has become a powerful tool for biomarker identification and target validation in drug discovery and biomedical research.

[0002] Currently available tissue arrayers on the market (including manual, semi-automatic, and automatic arrayers) typically use a pair of needles or tubular recipient block punches and donor block punches or samplers. In these tissue arrays, two basic steps are used to create a TMA block. 1) The recipient block punch makes small holes in the recipient TMA block, and 2) the donor block punch or sampler punches the donor tissue block to obtain tissue cores, which are then transferred and inserted into the small holes in the recipient block. The block is created by repeating the above two steps and is driven manually or automatically by a precision micrometer or other precision linear positioning mechanism.

[0003] Such tissue arrayers have many drawbacks, such as complex setup and operation, easy damage to the punches or paraffin blocks due to the impact of unprotected punching, uneven TMA tissue core surfaces due to variations in the height of the tissue cores on the surface, misalignment or distortion of the array or row lines of the tissue cores in the TMA block. In addition, these tissue arrayers driven by a micrometer or other mechanical positioning mechanism were unable to create frozen TMA blocks using frozen tissue. These drawbacks have greatly hindered the creation of high-quality TMA blocks and their wide application in the biomedical field.

[0004] There is a need to develop a tissue array layer that can solve the above-mentioned drawbacks.

Summary of the Invention

Means for Solving the Problems

[0005] In one embodiment of the present invention, there is provided an array layer for fabricating a tissue array, comprising a recipient block holder having a void for accommodating an upper surface and a tissue recipient block, and a guide plate that is structurally designed to be joined to the upper surface of the recipient block holder and includes a through-hole array aligned with the void of the recipient block holder. The recipient block holder and the guide plate are designed to be fixed to each other by a fixing assembly.

[0006] In some embodiments, the fixing assembly comprises a lock nut. The fixing assembly may further comprise a threaded hole on the guide plate.

[0007] In some embodiments, the void is provided with a stopper for restricting the position of the recipient block.

[0008] In some embodiments, the guide plate and the recipient block holder are repeatedly detachable, and when the guide plate and the recipient block holder are mounted together, the guide plate and the block holder are aligned at the same position.

[0009] The array layer may further comprise an LED light source disposed below the void. The LED light source may include a plurality of LED bulbs mounted on an LED substrate. The array layer may further comprise an LED light diffusing plate disposed above the LED light source and below the recipient block.

[0010] The through-hole array on the guide plate can be arranged such that the x-direction and the y-direction are perpendicular to each other.

[0011] The array layer further includes a positioning sliding member that is slidably connected to the guide plate and can move in a direction parallel to one of the x and y directions of the through holes.

[0012] The block holder and the guide plate of the array layer can be manufactured from a metal such as brass.

[0013] In another embodiment, provided is a full set for tissue array production, including the array layer described in this specification, a first punch pen for forming holes in a recipient block, and a second punch pen or sampling pen for punching out a tissue core from a tissue of interest and transferring it into the holes in the recipient block. The first punch pen and the second punch pen each include a hollow punch and a telescopic pin that can slide inside the hollow punch. Each hollow punch of the first and second punch pens can be manufactured from a metal such as brass.

[0014] In another embodiment, positioning a recipient block in a void of a recipient block holder, securing a guide plate in alignment with the recipient block holder, forming a hole in the recipient block by passing a punch of a recipient block punch pen through one of the through-holes of a through-hole array on the guide plate and extracting a paraffin core from the recipient block under the plate, inserting a donor block punch pen (or sampling pen) into a portion of interest of tissue within the donor block to obtain a tissue core and placing it in the punch hollow portion of the donor block punch pen, placing the punch of the donor block punch pen above one of the through-holes of the guide plate, and using the pin of the donor block punch pen to pass the donor tissue core of interest through one of the through-holes of the guide plate and press it into the hole formed on the recipient block. The LED light source can be turned on during the operation of the array layer. The donor tissue of interest may be frozen tissue or paraffin-embedded tissue. To obtain a frozen tissue sample, the collection can be performed in a low-temperature environment (temperature below 20 degrees Celsius). To obtain a paraffin-embedded tissue sample, the collection can be performed indoors or in the ambient environment.

[0015] In another embodiment, there is provided a method for fabricating a tissue array, comprising the steps of: preparing an array layer comprising (a) a recipient block holder having a void for accommodating an upper surface and a tissue recipient block, and (b) a guide plate structurally designed to be joined to the upper surface of the recipient block holder and including an array of through-holes aligned with the void of the recipient block holder; positioning the recipient block within the void of the recipient block holder; securing the guide plate to the recipient block holder; forming a hole in the recipient block positioned within the void by passing through one of the through-holes of the guide plate safely protected by the recipient block holder; harvesting an interested tissue core from a donor tissue; and transferring the interested tissue core into the hole formed on the recipient block.

[0016] The harvesting may include inserting the punch of a recipient block punch pen through one of the through-holes of the guide plate, punching out a paraffin core from the recipient block to form a cavity, and then pushing an interested tissue core within a donor block punch pen (or sampling pen) through one of the through-holes of the guide plate into the hole formed on the recipient block. The source of the tissue core may be paraffin-embedded tissue. The harvesting of the paraffin-embedded tissue core can be carried out at room temperature or ambient temperature. Alternatively, the source of the tissue core may be frozen tissue. In the case of frozen tissue, the harvesting of the tissue core can be carried out in a low-temperature environment.

[0017] The drawings, which are incorporated herein and form a part of the present specification, illustrate the apparatus and method of the present invention and are provided for further understanding. The drawings, together with the description, are used only to interpret some embodiments of the present invention and do not limit the scope of the present invention. In the drawings, like components are denoted by the same reference numerals.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides an array layer for a tissue array (or tissue microarray), a full set for producing a tissue array, and a method of using the same.

[0020] FIG. 1 shows an exploded view (left) of the tissue array layer components of the present invention and the assembled array layer (right). The meanings of the reference numerals used in the figure are as follows. 101 is a tissue array layer base, 102 is a recipient block holder, 103 is a guide plate, 104 is a positioning sliding member, 105 is an LED panel, 106 is a lock nut for a self-aligning plate, 107 is a battery chamber cover, 108 is an LED light diffusing plate, 109 is a battery chamber, 110 is a fixing screw for the positioning sliding member, 111 is a switch for the LED lamp, 112 is a switch nut for the LED panel, 113 is a battery, 114 is a screw for the battery chamber cover, 115 is a screw for joining the array layer base, 116 is a screw for the LED panel, and 117 represents a recipient block.

[0021] As shown in FIG. 1, the recipient block holder 102 includes two side portions whose upper surface 102a is coupled to the guide plate 103a via two threaded holes 102a1 and two lock nuts 106 for self-aligning plates, and a central portion having a reduced thickness, a top surface 102b, a central through hole 102d, and a blocking protrusion 102c disposed on one side. A void 102v is formed in the recipient block holder 102 and is structurally designed such that its size and dimensions can accommodate the recipient block 117, and the recipient block 117 can be slid in from the front side of the void. The horizontal position of the recipient block is fixed by the stopper 102c. The guide plate 103 is substantially flat and is provided at the top of the upper surface 102a of the recipient block holder 102. There is an array of through holes 103c aligned with the void of the recipient block holder 102 at the central portion of the guide plate 103. The recipient block holder 102a and the guide plate 103 are to be fixed by a fixing assembly. As shown in the figure, the guide plate 103 has two threaded holes 103a, and two self-aligning lock nuts 106 are inserted through the threaded holes 103a on the guide plate 103 and aligned with the threaded holes 102a1 to be connected to the upper surface 102a of the recipient block holder 102.

[0022] The recipient block holder 102 is coupled to the top of the array base 101 by a coupling screw 115, and the through hole 102d of the recipient block holder 102 is aligned with the hollow base 101a of the array base 101. The array base 101 includes an illumination assembly for the array and a power source for the illumination assembly. The illumination assembly includes an LED panel 105 (including an array of LED bulbs 105a) disposed under the hollow base 101a of the array base. The LED light of the LED bulbs transmits from the bottom and provides illumination for a generally partially translucent recipient block (e.g., a paraffin block) to facilitate operation of the array. The array base 101 also includes a battery holder 109 capable of accommodating a battery 113 and a battery cover 107 that can be tightly fixed to the battery holder by a screw 114. The LED bulbs are turned on and off by an LED switch 111. The LED light diffuser plate 108 can be disposed on the top of the LED panel 105, e.g., in a recess on the top of the hollow base 101a of the array base 101.

[0023] The array base 101 is separated from the recipient block holder by removing the coupling screw 115. The block holder and the guide plate combination can be operated without the array base 101 and the various components contained therein (e.g., forming a hole and pushing a tissue core into the hole). Such an operation is particularly advantageous when collecting a frozen tissue core and pushing it into the freezing chamber.

[0024] Figure 2 is a schematic view showing a guide plate. Cross-section A-A is a cross-sectional view of the guide plate having lock nut screw holes 103a at both ends, cross-section B-B is a cross-sectional view of the guide plate having array through-holes 103c, and Figure C is an enlarged cross-sectional view of the array through-hole having a chamfered top for easily and accurately receiving a donor block punch chip (see Figures 3 to 6 below). The lock nut 106 shown in Figure 1 comprises a tapered base that fits the top tapered portion of the screw hole 103a. The array through-holes are arranged in a matrix in the x and y directions (x is the length direction of the guide plate, and y is the width direction of the guide plate). Typical widths and lengths of the guide plate are 1.5 inches (38 mm) and 3 1 / 4 inches (82 mm), but other sizes can be selected as required. The thickness of the guide plate can be selected, for example, from 1 to 3 mm, or 1.5 to 2.5 mm, such as 1.5 mm, 2 mm, 2.5 mm, as required. The size of the through-hole on the plate is selected according to the application, and is, for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3.5 mm, 4.5 mm, etc.

[0025] Figure 3 is a cross-sectional view and an exploded view of the punch pen. Cross-section A-A shows the punch pen in the pressed position, and cross-section B-B shows the punch pen in the released position. In this figure, both the punch pen cap 224 and the punch pen cap lock nut are screwed onto the punch pen pressing rod 220 enclosed within the punch pen body 218. A punch pen pin 221 is connected to the distal end of the punch pen pressing rod 220, and the punch pen pin 221 has an enlarged portion 221a (proximal end of the coupling spring 222) near the proximal end. The body of the pin 221 passes through the center of the spring 222 and through the internal cavity of the punch 223 of the hollow punch pen having a distal tip 223a. When the spring 222 is released, the distal end of the pin retracts into the punch of the punch pen and becomes invisible from the outside. When the pen cap 224 is pushed to compress the spring 222, the distal end of the pin 222 protrudes from the distal tip 223a of the punch 223 of the punch pen, thereby pushing the sample collected in the hollow interior of the distal tip 223a just now. The punch of the punch pen is connected to the punch protector 219 of the punch pen.

[0026] To fabricate the TMA block, two punch pens (recipient block punch pen and donor block punch pen) can be used. The recipient block punch pen can be used to form holes in the recipient block (e.g., paraffin block), and the donor block punch pen is used to punch out tissue cores from the donor block and push the tissue cores through the guide plate into the holes formed in the recipient block. The diameter of the recipient block punch pen tip is slightly smaller than the diameter of the array through-holes of the guide plate, allowing the recipient block punch pen to form holes on the recipient block through the array through-holes. The diameter of the donor block punch pen tip is larger than the diameter of the array through-holes of the guide plate, but the diameter of the distal end of the donor block punch pen pin is smaller than the diameter of the array through-holes of the guide plate, enabling the donor block punch pen pin to push the tissue core punched out through the array through-holes of the guide plate into the holes formed on the recipient block.

[0027] Figure 4 shows an example of the step of forming holes in the recipient block by passing the recipient block punch pen through the guide plate.

[0028] Step 1: Place the recipient block 117 into the recipient block holder 102, and slightly tighten the lock nut 106 for self-aligning the guide plate. Turn on the LED panel 105 covered by the block holder 102 by the switch 111 (the nut 112 is used to fix the switch 111). Move the positioning sliding member 104 (fixed on the positioning sliding member 104 by the nut 110), and fix it in front of the first row of the array on the guide plate, and use the positioning sliding member 104 as the general starting position of each column. The positioning sliding member 104 has two distal ends joined to the edge of the guide plate and can move in the length direction of the guide plate.

[0029] Step 2: With the cap 224 in the release position, insert the punch 223 of the recipient block punch pen, pass it through the array through holes on the guide plate 103, and push it into the recipient block 117. The wide openings at the top of each threaded hole on the guide plate receive the punch of the punch pen and automatically align. The hole (or plate) wall has sufficient thickness to allow the punch to move vertically within the recipient block and form a vertical hole. The operator should also hold the punch pen as vertically as possible in alignment with the threaded holes on the guide plate. Cross-section A - A is a cross-sectional view of the tissue array layer of the present invention for forming holes in the recipient block, Figure B is an enlarged cross-sectional view of the recipient block punch entering the recipient block through the hole of the guide plate, and Figure C is a photo of the recipient block punch pen entering the recipient block 117 through the guide plate.

[0030] Step 3: Remove the recipient block punch from the recipient block, and press the cap of the recipient block punch pen to extrude the paraffin core from the recipient block. Figure D is a photograph showing the recipient block punch pen extruding the paraffin core from the recipient block, and Figure E shows how the hole in the recipient block can be confirmed through the guide plate under the LED light.

[0031] Figure 5 shows an example of the step of the donor block punch pen extracting the tissue core from the donor block (e.g., tissue sample). For convenience, the reference numerals of the components of the donor block punch pen are the same as those of the components of the recipient block punch pen.

[0032] Step 1: Adjust the donor block punch pen cap 224 up and down according to the thickness of the recipient block and the donor tissue to reach the appropriate depth of the punch pen pin 221, and tighten the cap type lock nut 225.

[0033] Step 2: With the punch pen cap 224 in the released position, press the punch 223 of the donor block punch pen into the selected point on the donor block 117a. Cross-section A-A is a cross-sectional view when the donor block punch penetrates and enters the selected block on the donor block, Figure B is an enlarged cross-sectional view when the donor block punch is inside the donor block and the donor tissue core is inside the punch, and Figure C is a photograph showing the donor block punch pen penetrating and entering the donor block 117a. To prevent damage such as cracking of the donor block due to the impact of punching, place a guide plate with an array of through holes (as shown and described in this specification) on the top of the donor block, and the donor block 117a can be punched through the large array of through holes on the plate (see Photograph D).

[0034] The donor block punch pen facilitates operations on the donor block on a light box under a magnifying glass or dissection microscope, or is simply easy to operate on the donor block held by hand against light. Particularly in the case of thin tissue sections or small biopsies, in order to maximally extract the donor tissue core, the punch can be pushed into the donor block at any desired angle (see Photo E), and the punch can be moved along the surface or inside of the donor block to extract the required tissue core.

[0035] The donor block shown in Figure 5 is a paraffin block, but the punch pen can also be used to extract frozen tissue cores in a similar manner. The extraction and transfer of frozen tissue sample cores using the donor block punch pen can be carried out in a low-temperature environment (temperature of -20°C or lower) such as a laboratory freezer or a dry freezer. The array layer (regardless of the presence or absence of the array base) can be brought into the low-temperature environment for operation.

[0036] Figure 6 shows an example of the step of inserting the donor tissue core through the guide plate into the recipient block.

[0037] Step 1: Remove the donor block punch pen from the donor block and place the punch of the pen at the top of the array through-hole on the guide plate (Figure C).

[0038] Step 2: Hold the donor block punch pen as vertically as possible, press the cap of the pen against the array through-hole on the guide plate 103, and push the donor tissue core into the recipient block 117. Cross-section A-A is a cross-sectional view of the donor block punch pen on the array through-hole of the guide plate in the state where the cap is pressed and the donor tissue core enters the recipient block. Figure B is an enlarged cross-sectional view of the donor block punch pushing the tissue core into the recipient block through the pin, Figure C is a view showing the donor block punch pen standing upright at the top of the array through-hole on the guide plate, and Figure D is a view showing the state where the tissue core inserted into the recipient block through the guide plate can be confirmed under the LED light.

[0039] FIG. 7 shows a photograph of an example of a TMA block produced by the apparatus and method of the present invention. FIG. A shows a high-density TMA block containing 228 tissue cores, and FIG. B shows an H&E stained TMA section of FIG. A.

[0040] Advantages of the embodiments of the present invention include ● The array layer of the present invention is low in cost and is affordable for producing TMA blocks with a wide range of tissue core sizes or densities.

[0041] ● Other available tissue array layers generally include precision parts for accurate movement and require special care and packaging for transportation or movement. In contrast, the array layer of the present invention can be manufactured in a very compact size and has shock resistance, making it easy to carry and operate.

[0042] ● Other available array layers generally require specific training, limited platforms, electricity, light, optical magnifiers, frequent calibration, etc. In contrast, the array layer of the present invention provides the flexibility to produce tissue array blocks in any environment. The simple structure and assembly of the array layer of the present invention enable mass production of TMA blocks simultaneously using multiple array layers within a limited space, which cannot be achieved by conventional TMA array layers.

[0043] ● The device is an automatic alignment guide plate that provides a traceable position for each tissue core on the recipient block holder. This feature enables accurate repair, refilling, or replacement of the tissue cores within the TMA block after TMA block production.

[0044] ● Both the guide plate and the recipient block holder can be made of metal (e.g., brass) and can be separated from the array base. The punch pen or components (e.g., punches and / or pins) can be made of metal (e.g., stainless steel). Therefore, due to their small size, the guide plate and the recipient block holder can be easily stored in a low-temperature environment such as a freezer chamber used for making boxes filled with dry ice or frozen tissue array blocks.

[0045] ● Note that the array layer of the present invention can produce a high-quality TMA block with minimal impact damage to the recipient TMA block, having a uniform tissue core surface and accurate tissue core alignment.

[0046] As described above, the embodiments for carrying out the present invention have been explained, but the present invention is not limited to the specific forms defined. These should be understood as substitutions, modifications, and equivalent variations within the scope of the present invention. Also, the present invention includes other embodiments that will be apparent to those skilled in the art.

Claims

1. A recipient block holder having an upper surface and a void, the void comprising a central portion configured to receive a recipient block; A guide plate configured to be connected to the upper surface of the recipient block holder and including a through-hole array aligned with the void of the recipient block holder; An array layer for fabricating a tissue array, comprising: The recipient block holder and the guide plate are fixed to each other by a fixing assembly, the array layer.

2. The fixing assembly includes a lock nut that enables the first exact positioning between the guide plate, the recipient block, and the recipient block holder to be reliably restored at any time. The array layer according to claim 1.

3. The guide plate includes a plurality of threaded holes. The array layer according to claim 2.

4. A stopper for restricting the position of the recipient block is provided in the void. The array layer according to claim 1.

5. The guide plate and the recipient block holder can be repeatedly and safely attached and detached, and when the guide plate and the recipient block holder are safely fixed, the guide plate and the recipient block holder are aligned at the same position. The array layer according to claim 1.

6. The array layer according to claim 1, further comprising an LED light source disposed below the void.

7. The LED light source includes a plurality of LED bulbs mounted on an LED substrate. The array layer according to claim 6.

8. The array layer according to claim 6, further comprising an LED light diffusion plate disposed above the LED light source.

9. The through-hole array on the guide plate is arranged such that the x-direction and the y-direction are perpendicular to each other. The array layer according to claim 1.

10. The array layer according to claim 1, further comprising a positioning sliding member slidably connected to the guide plate and movable in a direction parallel to one of the x and y directions of the through-hole.

11. The recipient block holder and the guide plate are made of metal. The array layer according to claim 1.

12. Examples of the metal include brass. The array layer according to claim 11.

13. The array layer according to claim 1, and A first punch pen for forming holes in a recipient block, and a second punch pen for transferring the tissue core of an organization of interest into the holes in the recipient block A full set for producing a tissue array, comprising: The first punch pen and the second punch pen each comprise a hollow punch and a telescopic pin that can slide within the hollow punch. A full set.

14. The full set according to claim 13, wherein the hollow punches of the first punch pen and the second punch pen are made of metal.

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