Plate-guided 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 technology]

[0001] Tissue arrays or tissue microarrays (TMA) usually refer to recipient blocks that contain hundreds of individual cylindrical tissue cores arrayed together. Each of these recipient blocks can be cut into hundreds of thin sections (TMA tissue sections) that 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 (including manual, semi-automated and automated arrayers) on the market typically use a pair of needle or tubular recipient block punches and donor block punches or samplers. These tissue arrays use two basic steps to create a TMA block: 1) the recipient block punch punches small holes in the recipient TMA block, and 2) the donor block punch or sampler pierces the donor tissue block to obtain tissue cores, which are then transferred and inserted into the small holes in the recipient block. The above two steps are repeated to create a block, which is driven manually or automatically by a precision micrometer or other precise linear positioning mechanism.

[0003] Such tissue arrayers have many drawbacks, such as complicated setup and operation, easy damage to the punch or paraffin block due to unprotected punch impact, uneven TMA tissue core surface due to variation in height of tissue cores on the surface, misaligned or distorted array or row lines of tissue cores in the TMA block, etc. Also, these tissue arrayers driven by micrometer or other mechanical positioning mechanisms could not use frozen tissue to create frozen TMA blocks. These drawbacks have greatly hindered the creation of high-quality TMA blocks and their widespread application in the biomedical field.

[0004] There is a need to develop a tissue arrayer that can overcome the above drawbacks. Summary of the Invention [Means for solving the problem]

[0005] In one embodiment of the present invention, an arrayer for producing tissue arrays is provided, comprising a recipient block holder having an upper surface and a cavity for receiving a tissue recipient block, and 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 cavity of the recipient block holder, the recipient block holder and the guide plate being designed to be fixed to each other with a fixing assembly.

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

[0007] In some embodiments, the gap is provided with a stop to limit 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 attached together, the guide plate and the block holder are aligned in the same position.

[0009] The arrayer may further comprise an LED light source disposed below the gap. The LED light source may include a plurality of LED bulbs mounted on an LED substrate. The arrayer may further comprise an LED light diffuser disposed above the LED light source and below the recipient block.

[0010] The array of through-holes on the guide plate can be arranged such that the x and y directions are orthogonal to each other.

[0011] The arrayer further comprises a positioning slide member slidably coupled to the guide plate and movable in a direction parallel to one of the x and y directions of the through holes.

[0012] The arrayer block holder and guide plates may be made of a metal such as brass.

[0013] In another embodiment, a full set for making tissue arrays is provided, comprising an arrayer as described herein, a first punch pen for forming holes in a recipient block, and a second punch or sampling pen for cutting tissue cores from tissues of interest and transferring them to holes in the recipient block. The first punch pen and the second punch pen each comprise a hollow punch and an extendable pin that can slide within the hollow punch. Each hollow punch of the first and second punch pens can be made of a metal such as brass.

[0014] In another embodiment, a method of operating the arrayer described herein is provided, comprising the steps of: positioning a recipient block in a gap of a recipient block holder; fitting and fixing a guide plate to the recipient block holder; passing a punch of a recipient block punch pen through one of the holes of a hole array on the guide plate to hollow a paraffin core from the recipient block below the plate, forming a hole in the recipient block; inserting a donor block punch pen (or sampling pen) into a portion of tissue of interest in the donor block to obtain a tissue core and placing it in the punch hollow portion of the donor block punch pen; placing a punch of the donor block punch pen over one of the holes of the guide plate and using a pin of the donor block punch pen to push a donor tissue core of interest through one of the holes of the guide plate into the formed hole on the recipient block. The LED light source can be turned on during operation of the arrayer. The donor tissue of interest can be a frozen tissue or a paraffin-embedded tissue. To obtain frozen tissue samples, collection can be performed in a cryogenic environment (temperature below 20 degrees Celsius). To obtain paraffin-embedded tissue samples, collection can be performed in room or ambient environment.

[0015] In another embodiment, a method for making a tissue array is provided, comprising the steps of: providing an arrayer comprising: (a) a recipient block holder having an upper surface and a cavity for receiving a tissue recipient block; and (b) a guide plate structurally designed to mate with the upper surface of the recipient block holder and including an array of through holes aligned with the cavity of the recipient block holder; positioning a recipient block within the cavity of the recipient block holder; fitting and securing the guide plate to the recipient block holder; forming a hole in the recipient block through one of the through holes of the guide plate secured by the recipient block holder and positioned within the cavity; harvesting a tissue core of interest from a donor tissue; and transferring the tissue core of interest into the hole formed on the recipient block.

[0016] The harvesting may include inserting a punch of a recipient block punch pen through one of the holes in the guide plate to excavate a paraffin core from the recipient block to form a hole, and then forcing a tissue core of interest in a donor block punch pen (or sampling pen) through one of the holes in 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 may be performed at room 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 may be performed in a cryogenic environment.

[0017] The drawings are incorporated in and constitute a part of this specification to illustrate and provide a further understanding of the apparatus and method of the present invention. The drawings together with the description are used only to interpret some embodiments of the present invention, but are not intended to limit the scope of the present invention. In the drawings, similar elements are designated by the same reference numerals. [Brief description of the drawings]

[0018] [Figure 1] FIG. 2 shows an exploded view of some components of an arrayer according to an embodiment of the present invention and an assembled arrayer. [Diagram 2] 3 is a schematic diagram of a guide plate according to an embodiment of the present invention. FIG. [Diagram 3] 1A and 1B are a cross-sectional view and an exploded view of a punch pen according to an embodiment of the present invention. [Figure 4] 1A-1C are diagrams illustrating an example of a step in which a recipient block punch pen according to an embodiment of the present invention forms a hole in a recipient block. [Diagram 5] 1A-1C show an example of a step in which a donor block punch pen according to an embodiment of the present invention punches a tissue core from a donor block. [Figure 6] 13A-13C show an example of a step of inserting a donor tissue core into a recipient block through a guide plate according to an embodiment of the present invention. [Figure 7] 1 is a photograph of an example of a TMA block produced by an apparatus and method according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention provides an arrayer for tissue arrays (or tissue microarrays), a full set for producing tissue arrays, and methods of using the same.

[0020] Figure 1 shows an exploded view of the tissue arrayer components of the present invention (left) and an assembled arrayer (right). The symbols used in the figure have the following meanings: 101 is the tissue arrayer base, 102 is the recipient block holder, 103 is the guide plate, 104 is the positioning slide, 105 is the LED panel, 106 is the lock nut for the self-aligning plate, 107 is the battery chamber cover, 108 is the LED light diffuser, 109 is the battery chamber, 110 is the fixing screw for the positioning slide, 111 is the switch for the LED light, 112 is the switch nut for the LED panel, 113 is the battery, 114 is the screw for the battery chamber cover, 115 is the screw for connecting the arrayer base, 116 is the screw for the LED panel, and 117 is the recipient block.

[0021] As shown in FIG. 1, the recipient block holder 102 has two sides with a top surface 102a connected to the guide plate 103a via two threaded holes 102a1 and two lock nuts 106 for self-aligning plates, and a central portion with a reduced thickness, a top surface 102b, a central through hole 102d and a blocking protrusion 102c located on one side. The recipient block holder 102 is formed with a cavity 102v whose size and dimensions are structurally designed to accommodate the recipient block 117, and the recipient block 117 can slide into the cavity from the front side. The horizontal position of the recipient block is fixed by a stopper 102c. The guide plate 103 is substantially flat and is provided on top of the top surface 102a of the recipient block holder 102. In the center of the guide plate 103, there is an array of through holes 103c aligned with the cavity of the recipient block holder 102. The recipient block holder 102a and the guide plate 103 are 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 connect to the upper surface 102a of the recipient block holder 102.

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

[0023] The arrayer base 101 is separated from the recipient block holder by removing the connecting screws 115. The block holder and guide plate combination can be manipulated (e.g., drilling holes and forcing tissue cores into the holes) without the arrayer base 101 and the various components contained therein. Such manipulation is particularly advantageous when harvesting frozen tissue cores and forcing them into the freezing chamber.

[0024] FIG. 2 is a schematic diagram showing a guide plate. Section AA is a cross-sectional view of a guide plate with lock nut threading holes 103a at both ends, section BB is a cross-sectional view of a guide plate with array through holes 103c, and section C is an enlarged cross-sectional view of an array through hole with a chamfered top for easy and accurate reception of a donor block punch tip (see FIGS. 3-6 below). Lock nut 106 shown in FIG. 1 has a tapered base that fits the tapered top of threading 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 needed. The thickness of the guide plate can be selected as needed, for example, 1-3 mm, or 1.5-2.5 mm, for example, 1.5 mm, 2 mm, 2.5 mm. The size of the holes on the plate is selected depending on the application, for example 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3.5 mm, 4.5 mm, etc.

[0025] FIG. 3 shows a cross-sectional view and an exploded view of the punch pen. Section AA shows the punch pen in the pressed position, and section BB shows the punch pen in the released position. In this view, the punch pen cap 224 and the punch pen cap lock nut are both screwed onto the punch pen pressing rod 220 enclosed in 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 (the 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 passes through the internal cavity of the hollow punch pen punch 223 with the distal tip 223a. When the spring 222 is released, the distal end of the pin retracts into the punch pen punch and is not visible from the outside. When the pen cap 224 is pressed to compress the spring 222, the distal end of the pin 222 is protruded from the distal tip 223a of the punch pen punch 223, thereby forcing the sample previously collected into the hollow interior of the distal tip 223a. The punch pen punch is connected to the punch pen punch protector 219.

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

[0027] FIG. 4 shows an example of the steps of passing a recipient block punch pen through a guide plate to form holes in the recipient block.

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

[0029] Step 2: With cap 224 in the release position, insert the punch 223 of the recipient block punch pen through the array through hole on the guide plate 103 and push it into the recipient block 117. A wide opening at the top of each threaded hole on the guide plate accepts and automatically aligns the punch of the punch pen. The hole (or plate) wall is thick enough to allow the punch to move vertically into the recipient block and form a vertical hole. The operator also holds the punch pen as vertically as possible to align it with the threaded hole on the guide plate. Section AA is a cross-sectional view of the tissue arrayer of the present invention for forming a hole in the recipient block, view B is an enlarged cross-sectional view of the recipient block punch that entered into the recipient block through the hole in the guide plate, and view C is a photograph of the recipient block punch pen that entered into the recipient block 117 through the guide plate.

[0030] Step 3: Take out the recipient block punch from the recipient block, and press the cap of the recipient block punch pen to push the paraffin core out of the recipient block. Figure D is a photo of the recipient block punch pen pushing the paraffin core out of the recipient block, and Figure E shows that under the LED light, the hole in the recipient block can be seen through the guide plate.

[0031] 5 shows an example of a donor block punch pen punching a tissue core from a donor block (e.g., a tissue sample). For convenience, the components of the donor block punch pen are numbered the same as those of the recipient block punch pen.

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

[0033] Step 2: With punch pen cap 224 in the released position, push punch 223 of the donor block punch pen into the selected point on donor block 117a. Section AA is a cross section of the donor block punch as it penetrates into the selected portion of the donor block, section B is a close-up cross section of the donor block punch in the donor block and with the donor tissue core inside the punch, and section C is a photograph of the donor block punch pen as it penetrates into donor block 117a. To prevent damage such as cracking of the donor block due to the impact of the punching, a guide plate with an array of holes (as shown and described herein) can be placed on top of the donor block and punches can be made into the donor block 117a through the large array of holes on the plate (see photo D).

[0034] The donor block punch pen allows easy manipulation of the donor block on a light box under a magnifying glass or dissecting microscope, or simply on a donor block held up to the light by hand. To maximize the amount of donor tissue core excised, especially for thin tissue sections or small biopsies, the punch can be pressed into the donor block at any desired angle (see photo E) and the punch can be moved along the surface or interior of the donor block to excis the desired tissue core.

[0035] The donor block shown in Figure 5 is a paraffin block, but a punch pen can be used to cut frozen tissue cores in a similar manner. Cutting and transferring frozen tissue sample cores with a donor block punch pen can be performed in a cold environment (temperature below -20°C) such as a laboratory freezer or dry freezer. The arrayer (with or without an arrayer base) can be brought into the cold environment and operated on.

[0036] FIG. 6 illustrates an example of the steps of inserting a donor tissue core through a guide plate and into a recipient block.

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

[0038] Step 2: Holding the donor block punch pen as vertically as possible, press the cap 224 of the pen into the array holes on the guide plate 103 to push the donor tissue cores into the recipient block 117. Section AA is a cross-sectional view of the donor block punch pen over the array holes of the guide plate with the cap pressed and the donor tissue cores in the recipient block. Fig. B is an enlarged cross-sectional view of the donor block punch pushing the tissue cores into the recipient block via the pins, Fig. C shows the donor block punch pen standing upright on top of the array holes on the guide plate, and Fig. D shows how the tissue cores inserted into the recipient block through the guide plate can be seen under LED light.

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

[0040] Advantages of embodiments of the present invention include: • The arrayer of the present invention is low cost and affordable for producing TMA blocks of a wide range of tissue core sizes or densities.

[0041] Other available tissue arrayers typically contain precision parts for precise movement and require special care and packaging for transportation or movement. In contrast, the arrayer of the present invention can be manufactured in a very compact size and is impact resistant, making it easy to carry and operate.

[0042] ●Other available arrayers generally require specific training, limited platform, electricity, light, optical magnification, frequent calibration, etc. In contrast, the arrayer of the present invention provides the flexibility to create tissue array blocks in any environment. The simple structure and assembly of the arrayer of the present invention allows for simultaneous mass production of TMA blocks using multiple arrayers in a limited space, which is something that conventional TMA arrayers cannot achieve.

[0043] The device is an auto-aligning guide plate that provides a trackable position of each tissue core on the recipient block holder. This feature allows for precise repair, refilling or replacement of tissue cores in a TMA block after TMA block creation.

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

[0045] Furthermore, the arrayer of the present invention can minimize impact damage to the recipient TMA block and produce high-quality TMA blocks with uniform tissue core surfaces and precise tissue core alignment.

[0046] Although the embodiments for carrying out the present invention have been described above, the present invention is not limited to the specific embodiments defined above. These should be understood as replacements, modifications, and equivalent variations within the scope of the present invention. The present invention also includes other embodiments that are obvious to those skilled in the art.

Claims

1. a recipient block holder having an upper surface and a cavity for receiving a tissue recipient block; a guide plate designed to mate with an upper surface of the recipient block holder and including an array of through holes aligned with the apertures of the recipient block holder; An arrayer for producing a tissue array, comprising: An arrayer, in which the recipient block holder and the guide plate are designed so that the height of both gaps can be adjusted to accommodate recipient blocks of different thicknesses or different sizes, the recipient block is ensured to return to the correct position even after being moved, and the fixing assembly allows the recipient block to be safely protected during multi-directional movement or transportation of the arrayer.

2. 2. The arrayer of claim 1, wherein the fixation assembly includes a lock nut that ensures return to an initial, correct position between the guide plate, the recipient block, and the recipient block holder at all times.

3. The arrayer of claim 2 , wherein the guide plate includes a plurality of threaded holes.

4. The arrayer of claim 1 , wherein the gap is provided with a stopper that limits the position of the recipient block.

5. 2. The arrayer of claim 1, wherein the guide plate and the recipient block holder can be safely attached and detached repeatedly, and when the guide plate and the recipient block holder are safely secured, the guide plate and the recipient block holder align in the same position.

6. The arrayer of claim 1 further comprising an LED light source disposed below the gap.

7. The arrayer of claim 6 , wherein the LED light source comprises a plurality of LED bulbs mounted on an LED substrate.

8. The arrayer of claim 6 , further comprising an LED light diffuser disposed above the LED light sources.

9. The arrayer of claim 1 , wherein the array of through-holes on the guide plate is arranged such that the x- and y-directions are mutually orthogonal.

10. 2. The arrayer of claim 1, further comprising a positioning slide member slidably coupled to said guide plate and movable in a direction parallel to one of the x and y directions of said through holes.

11. The arrayer of claim 1 , wherein the recipient block holder and the guide plate are made of metal.

12. The arrayer of claim 11 , wherein the metal includes brass.

13. An arrayer according to claim 1; a first punch pen for forming a hole in the recipient block; a second punch pen for transferring a tissue core of a tissue of interest into the hole in the recipient block; A full set for producing a tissue array comprising: The first punch pen and the second punch pen are a full set including a hollow punch and an extendable pin that can slide within the hollow punch.

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.

15. A method for operating the arrayer according to any one of claims 1 to 11, comprising the steps of: positioning a recipient block in a cavity of a recipient block holder; aligning and securing a guide plate to the recipient block holder; forming a hole on the recipient block by inserting a punch of a recipient block punch pen through one of the through holes of the guide plate and punching a flat straight core from the recipient block; taking a tissue core of the tissue of interest on the donor block in a hollow punch of a donor block punch pen; applying pressure while positioning a punch of the donor block punch pen over one of the holes in the guide plate to drive a pin of the donor block punch pen through one of the holes in the guide plate and to force a tissue core of interest through one of the holes in the guide plate and into a hole formed on the recipient block; A method comprising:

16. The method of claim 15 , further comprising turning on the LED light source during operation of the arrayer.

17. 16. The method of claim 15, wherein obtaining a portion of the tissue of interest comprises obtaining a portion of frozen tissue.

18. 20. The method of claim 17, wherein collecting the tissue core of interest in the hollow punch of the donor block punch pen is performed in a cryogenic environment.

19. 16. The method of claim 15, wherein obtaining the tissue core of interest comprises obtaining a paraffin-embedded tissue core.

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