WO2025086267A1

WO2025086267A1 - Dynamically controlled immunohistochemistry (ihc) stainer systems and adjustable angle method

Info

Publication number: WO2025086267A1
Application number: PCT/CN2023/127243
Authority: WO
Inventors: Jee Jong Shum
Original assignee: Shenzhen Prs Ltd
Current assignee: Shenzhen Prs Ltd
Priority date: 2023-10-27
Filing date: 2023-10-27
Publication date: 2025-05-01
Anticipated expiration: 2026-04-27

Abstract

A staining system (100) includes a dynamically controlled external air pressurized heating chamber for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, the heating chamber of low mass and capable to be pressurized by external air. The staining system (100) includes an adjustable air pressure control mechanism (130). The staining system (100) further includes a capillary gap cover (102) that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool (114b). The staining system (100) further includes a submersion system (128) that submerges a slide (114a) within the buffer pool (114b), the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi. The staining system (100) further includes the heating chamber and the associated components are capable of being rotated so as to uniformly mix a reagent and a buffer.

Description

DYNAMICALLY CONTROLLED IMMUNOHISTOCHEMISTRY (IHC) STAINER SYSTEMS AND ADJUSTABLE ANGLE METHOD

TECHNICAL FIELD

This description relates to immunohistochemistry (IHC) staining and associated systems, devices, methods, and apparatuses including a system of an adjustable angle rocking action mechanism that improves the performance and efficiency of automated staining processes by achieving optimal mixing of buffers and reagents.

BACKGROUND

Stationary automated stainers do not perform well in distributing a staining solution throughout a tissue sample, and at times result in non-uniform staining, and non-uniform staining contributes to false indications in tissue analysis, for example, overly concentrated stain or faint stain indicating cancerous tissue.

SUMMARY

The present disclosure relates to an adjustable angle rocking action which uses gravity to perform a smooth and gentle mixing of the buffer or reagent by turning the chamber + and -8° slowly multiple times which is able to refresh the heat induced epitope retrieval (HIER) buffer inside the capillary gap, and also washes off the excess formalin so as to expose the maximum number of antigen sites.

A Legend 6G Stainer includes a dynamically controlled external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, said heating chamber being of low mass and capable of being pressurized by external air. The Legend 6G Stainer further includes an adjustable air pressure control mechanism that regulates the pressurization of the heating chamber. The Legend 6G Stainer further includes a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool. The Legend 6G Stainer further includes a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling, the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements. The Legend 6G Stainer further includes that heating chamber and the associated components are capable of being rotated so as to uniformly mix a reagent and a buffer.

A staining system includes a dynamically controlled external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, said heating chamber being of low mass and capable of being pressurized by external air. The staining system further includes an adjustable air pressure control mechanism configured to regulate the pressurization of the heating chamber. The staining system further includes a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool. The staining system further includes a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling, the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements. The Legend 6G Stainer further includes that heating chamber and the associated components are capable of being rotated so as to uniformly mix a reagent and a buffer.

A staining method comprising dynamically controlling an external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, said heating chamber being of low mass and capable of being pressurized by external air, regulating the pressurization of the heating chamber using an adjustable air pressure control mechanism, ensuring even heat distribution within a buffer pool using a capillary gap cover that protects a tissue during the HIER IHC staining process, submerging a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling using a submersion system, allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements using the heating chamber and associated components, and rotating the heating chamber and the associated components so as to uniformly mix a reagent and a buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the following detailed description when read with the accompanying figures. It is noted that, in accordance with the standard practice in the industry, various features are not drawn to scale. In fact, the dimensions of the various features may be arbitrarily increased or reduced for clarity of discussion.

Figure 1 is a schematic diagram of a stainer system, in accordance with some embodiments.

Figure 2 is a block diagram of system architecture in accordance with some embodiments.

Figure 3 is a sequence diagram of a method of rotating the stainer system in accordance with some embodiments.

Figure 4 is a cross section view of the rotating stainer system in accordance with some embodiments.

DETAILED DESCRIPTION

The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components, values, operations, materials, arrangements, or the like, are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. Other components, values, operations, materials, arrangements, or the like, are contemplated. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.

For the past 80 years, the application of heat in the Heat Induced Epitope Retrieval (HIER) process for immunohistochemistry (IHC) staining has been a challenging endeavor. Various methods, ranging from coplin jars to modern auto-stainers, have been explored to improve the efficiency and effectiveness of antigen retrieval. However, these methods often encounter limitations and drawbacks. Cold enzyme digestion processes have shown to be problematic as these processes damage numerous antigen sites. High heat antigen retrieval processes using pressure cookers at 120℃ are prone to inconsistencies. Most auto-stainers today apply temperatures approaching the boiling point which poses the risk of micro bubble formation and tissue damage. Existing HIER-based solutions suffer from heat-induced thermal flow, leading to biomaterial displacement and non-uniform antigen retrieval. The other systems uneven heating causes bubble formation and cell cluster detachment, while in other systems an air knife displaces reagents and results in the loss of cell features. The capillary gap, resembling a clam, also contributes to material loss from sectioned cells.

In addition to uneven heating, uneven or non-uniform staining of tissue samples presents numerous challenges in performing reliable tissue analysis. The Leica system uses a pump to suck liquid back and forth for mixing, the pump being too aggressive, resulting occasionally in folding of a tissue on the top and bottom. The Ventana system uses an air knife to displace reagents and mixing. This aggressive mixing and washing scavenges unknown amounts of materials out of sectioned cells, which leads to loss of the cell’s cytoplasm and nucleus features. The Dako capillary gap acts like a clam by opening and closing to move reagents about the tissue, however, the opening (pull force) is very hard on tissues because this pulling force sucks materials out of the sectioned cells.

Other devices, approaches and/or methods utilizing a pressure/rice cooker, and double steamer, as referred to above, apply heat from the bottom, and the slide, which is held vertically, suffers from heat induced thermal flow of the buffer. This flow causes dislocation of biomaterials, as well as non-uniformity in the antigen retrieval. This includes a closed capillary gap held in a fixed angle like the Sakura and Leica’s clone systems.

Figure 1 is a schematic diagram of a stainer system 100 (hereinafter referred to as “stainer system 100” ) , in accordance with some embodiments. Figure 1 is simplified for the purpose of illustration. The stainer system 100 is capable of carrying out, or being used for, one or more staining processes or procedures. See the below features also in the cross-section view of Figure 4.

The stainer system 100 includes a cover 102. In some embodiments, the cover 102 comprises a material suitable for forming an air-tight enclosure. In some embodiments, the cover 102 comprises a hard plastic or metallic material. In some embodiments, the cover 102 comprises a plexi-glass material. In some embodiments, the cover 102 comprises a polypropylene material. In some embodiments, the cover 102 comprises a polypropylene and polyethylene blend material. In some embodiments, the cover 102 comprises an acrylic material. In some embodiments, the cover 102 comprises another material.

The stainer system 100 includes a container 104. In some embodiments, the container 104 comprises a material suitable for forming an air-tight enclosure. In some embodiments, the container 104 comprises a high temperature hard plastic material. In some embodiments, the container 104 comprises a plexi-glass metallic material. In some embodiments, the container 104 comprises a Teflon coating material. In some embodiments, the container 104 comprises a polypropylene material. In some embodiments, the container 104 comprises a polypropylene and polyethylene blend material. In some embodiments, the container 104 comprises an acrylic material. In some embodiments, the container 104 comprises another material.

In some embodiments, the cover 102 and the container 104 are comprised of the same material. In some embodiments, the cover 102 and the container 104 are comprised of different materials.

The stainer system 100 includes a hinge 106 attached to the cover 102 and the container 104. In some embodiments, the hinge 106 allows for opening and closing of the cover 102. In some embodiments, the cover 102 and the container 104 are attached by more than one hinge or a hinge alternative. In some embodiments, the hinge 106 comprises a metallic material. In some embodiments, the hinge 106 comprises a plastic material. In some embodiments, the hinge 106 comprises a material that is the same material as the cover 102 or the container 104. In some embodiments, a hinge is not used.

In some embodiments, the container 104 forms a chamber area. In some embodiments, the chamber area is a heating chamber of low mass capable of being pressurized by external air or air from sources external to the chamber area, such as an air tank compressor, etc. In some embodiments, the chamber area includes an air-circulating section or portion for circulating air. In some embodiments, the air is heated. In some embodiments, the chamber area is an air-heating area or portion. In some embodiments, the heating chamber area and associated components allow users to adjust a pressure within a psi range, such as a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements. In some the cover 102 and the container 104 form an air-tight enclosure. In some embodiments, the cover 102 and the container 104 are capable of forming an air-tight seal.

The stainer system 100 includes a fan 108. In some embodiments, the fan is suitable for circulating air within a chamber area, causing the temperature of the air to equilibrate or become more consistent. In some embodiments, the fan 108 comprises an electrical fan. In some embodiments, the fan 108 is enclosed, shielded, or substantially enclosed or shielded. In some embodiments, the fan 108 is in communication with one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the fan 108 speed is capable of being controlled by the one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the fan 108 speed is adjusted based on a temperature of a chamber area. In some embodiments, the fan 108 speed is adjusted based on a temperature of dewax process. A buffer liquid, as described herein.

The stainer system 100 includes a heater element 110. In some embodiments, the heater element is suitable for heating or increasing the temperature of the air HIER buffer in a chamber area. In some embodiments, the heater element 110 comprises an electrical heater. In some embodiments, the heater element 110 is enclosed, shielded, or substantially enclosed or shielded. In some embodiments, the heater element 110 is in communication with one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the heater element 110 temperature is capable of being controlled by the one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the heater element 110 temperature is adjusted based on a temperature of a 114 chamber area. In some embodiments, the heater element 110 temperature is adjusted based on a temperature of a buffer liquid, as described herein.

The stainer system 100 includes an air inlet/outlet 112. In some embodiments, the air inlet/outlet 112 forms an air-tight passage into a chamber area. In some embodiments, the air inlet/outlet 112 is capable of adding or removing air from a chamber area. In some embodiments, the air inlet/outlet 112 is connected to an air pump or similar device. In some embodiments, the air inlet/outlet 112 is in communication with one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the air pressure in a chamber area is capable of being controlled by the one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the air pressure is controlled by an adjustable air pressure control mechanism 130. In some embodiments, the air pressure in a chamber area is adjusted based on a temperature of a chamber area. In some embodiments, the air pressure in a chamber area is adjusted based on a temperature of a buffer area, as described herein. In some embodiments, the adjustable air pressure control mechanism 130 comprises an air pressure measurement gauge or measurement device connected to a valve. In some embodiments, the valve is opened or closed to allow for air pressure adjustment.

The stainer system 100 includes a seating area 114. In some embodiments, the seating area 114 is capable of, or suitable for, holding a slide 114a or IHC utensil for staining. In some embodiments, stack a slide over another in an angle, so the chamber is capable to hold more slides per each staining process. In some embodiments, the slide or IHC utensil for staining includes a capillary gap cover that protects a tissue during an HEIR IHC staining process to ensure even heat distribution within a buffer pool. In some embodiments, the seating area 114 is capable of, or suitable for, containing a liquid, such as a buffer liquid. In some embodiments, the seating area 114 is capable of, or suitable for, containing a buffer pool 114b. In some embodiments, the seating area 114 includes or comprises a submersion system 128 that submerges a slide within a buffer pool to eliminate formation of hot spots and micro nucleated boiling. In some embodiments, the seating area 114 comprises a material suitable for conducting heat. In some embodiments, the seating area 114 comprises a hard plastic metallic material prefer stainless steel. In some embodiments, the seating area 114 comprises a plexi-glass material. In some embodiments, the seating area 114 comprises a polypropylene material. In some embodiments, the seating area 114 comprises a polypropylene and polyethylene blend material. In some embodiments, the seating area 114 comprises an acrylic material. In some embodiments, the seating area 114 comprises another material. In some embodiments, the seating area 114 comprises a dark or colored material usable to block light. In some embodiments, a submersion system 128 comprises a dish, bowl, or other device capable of containing a liquid.

The slide 114a is in a flat orientation 120, hence the staining is limited to the top layer opposed to staining of sub-layers. The turning of the container 104 is able to achieve the maximum antigen retrieval process. By implementing the adjustable angle of rocking action, the slide 114a experiences a dynamic movement that facilitates optimal antigen retrieval. The controlled rocking motion ensures comprehensive mixing of the buffer throughout the capillary gap, allowing for consistent and efficient staining.

The adjustable angle rocking action is able to be further customized for different stages of the staining process to optimize results. In some embodiments, during the dewaxing process, where the tissue is still in a solid state, a higher angle, such as an approximately 30° downward position 120, is able to expedite the removal of wax and xylene.

In some embodiments, in subsequent steps, such as the ethanol, distilled water (DI) , and phosphate buffered saline (PBS) wash cycles, a lower angle of an approximately 15° downward position 124/126 provides sufficient time for thorough removal of xylene residues. During the incubation period, the slide 114a is in a horizontal position (0°) 120 to maintain ideal conditions for the staining process.

This innovative feature offers flexible settings, empowering users to experiment with different angles and timings to achieve the best staining results based on specific tissue types and staining requirements.

By introducing the adjustable angle rocking action for buffer and reagent mixing, the embodiments of the present disclosure provide numerous advantages to the field of automated staining. The embodiments in the present disclosure hold immense potential across various domains, including medical diagnostics, research, and pathology, where automated staining techniques play a crucial role in achieving accurate and reliable results.

The embodiments in the present disclosure have numerous advantages, including improved staining uniformity such that the controlled rocking motion ensures comprehensive mixing, resulting in more uniform staining outcomes; preservation of cellular features such that the gentle mixing process minimizes the extraction of cellular materials, preserving crucial cytoplasm and nucleus features; enhanced antigen retrieval such that optimal exposure of antigen sites is achieved, leading to improved staining efficiency; customizability such that the mechanism allows users to adjust the rocking angles for different stages of the staining process, enabling optimization and flexibility; and applicability such that embodiments are able to be integrated into various automated staining systems, catering to diverse domains such as medical diagnostics, research, and pathology.

In some embodiments, the seating area 114 is removable attached to the container 204. In some embodiments, the seating area 114 is cup-shaped, rectangularly and cup-shaped, or U-shaped, and removably attached to the container 104. In some embodiments, the seating area 114 allows for suspension of an IHC slide, device, or utensil above a chamber area, allowing for heat conduction from the chamber area, through the seating area 114 or through a buffer liquid or pool.

The stainer system 100 includes a liquid drain 116 from the seating area 114. The liquid drain 116 allows for removal of liquid, such as a liquid buffer, from the seating area 114.

The stainer system 100 includes a temperature sensor 118. In some embodiments, the temperature sensor 118 allows for determination of a temperature of a liquid such as a buffer liquid in which an IHC slide or device is immersed. In some embodiments, the temperature sensor 118 is in communication with one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the temperature of a liquid, such as a buffer liquid, is capable of being controlled by the one or more processors, controllers, microcontrollers, or circuits. In some embodiments, the temperature of a liquid, such as a buffer liquid, is adjusted based on a temperature of a chamber area, or vice versa.

In some embodiments, various systems, sub-systems, and components from the aforementioned are combined and in communication with the same controlling unit comprising one or more processors, controllers, microcontrollers, or circuits.

Signals and Measurements Intake

In some embodiments, temperatures are continuously monitored by one or more temperature sensors. In some embodiments, temperatures are determined at predetermined intervals by one or more temperature sensors. For example, temperatures may be determined by one or more temperature sensors in a buffer liquid, chamber area, etc.

In some embodiments, air pressures are continuously monitored by one or more pressure sensors. In some embodiments, air pressures are determined at predetermined intervals by one or more pressure sensors. For example, air pressures may be determined by one or more pressure sensors in a chamber area, etc.

The temperature and pressure readings and measurements from various sensors are delivered to a controlling unit, for example.

Dynamic Control and Adjustment

The controlling unit allows for synchronization, adjustment, and calibration of various system components based on various system environment measurements.

For example, when a temperature sensor determines a temperature of a buffer pool falls below, or increases beyond, a predetermined temperature range, one or more signals may be delivered by the controlling unit to a heating element to increase or decrease the temperature so as to adjust the temperature of the buffer pool.

Similarly, in some examples, when a pressure sensor determines a pressure of a chamber area falls below, or increases beyond, a predetermined pressure range, one or more signals may be delivered by the controlling unit to a compressor element or system to increase or decrease the air pressure by adding or removing air through an air inlet/outlet so as to adjust the air pressure of the chamber area.

In some examples, physical chemistry relationships between temperature and pressure are used to adjust environment conditions in a chamber area or buffer pool. For example, pressure adjustments are used to modify boiling points and chamber temperatures and humidities for example, and vice versa.

The systems and methods herein are usable to dynamically control environment conditions, such as the conditions of a heating chamber or buffer pool, during a staining process. By utilizing a dynamic control process, the systems and methods herein help to prevent tissue damage, bubble formation, and uneven mixing of reagent and buffer during staining.

Figure 2 is a block diagram of system architecture 200 in accordance with some embodiments.

System architecture 200 includes a hardware processor 202 and a non-transitory, computer readable storage medium 204 encoded with, i.e., storing, the computer program code 206, i.e., a set of executable instructions. Computer readable storage medium 204 is also encoded with instructions 207 for interfacing with external devices. The processor 202 is electrically coupled to the computer readable storage medium 204 via a bus 208. The processor 202 is also electrically coupled to an I/O interface 210 by bus 208. A network interface 212 is also electrically connected to the processor 202 via bus 208. Network interface 212 is connected to a network 214, so that processor 202 and computer readable storage medium 204 are capable of connecting to external elements via network 214. The processor 202 is configured to execute the computer program code 206 encoded in the computer readable storage medium 204 in order to cause system architecture 200 to be usable for performing a portion or all of the operations as described herein.

In some embodiments, the processor 202 is a central processing unit (CPU) , a multi-processor, a distributed processing system, an application specific integrated circuit (ASIC) , or a suitable processing unit.

In some embodiments, the computer readable storage medium 204 is an electronic, magnetic, optical, electromagnetic, infrared, or a semiconductor system (or apparatus or device) . For example, the computer readable storage medium 204 includes a semiconductor or solid-state memory, a magnetic tape, a removable computer diskette, a random access memory (RAM) , a read-only memory (ROM) , a rigid magnetic disk, or an optical disk. In some embodiments using optical disks, the computer readable storage medium 304 includes a compact disk-read only memory (CD-ROM) , a compact disk-read/write (CD-R/W) , or a digital video disc (DVD) .

In some embodiments, the storage medium 204 stores the computer program code 206 configured to cause system architecture 200 to perform a portion or all of the operations as described herein. In some embodiments, the storage medium 204 also stores information needed for performing a portion or all of the operations as described herein as well as information generated during performing a portion or all of the operations as described herein, such as a user interface parameter 316.

In some embodiments, the storage medium 204 stores instructions 207 for interfacing with external devices. The instructions 207 enable processor 202 to generate instructions readable by the external devices to effectively implement a portion or all of the operations as described herein.

System architecture 200 includes I/O interface 210. I/O interface 210 is coupled to external circuitry. In some embodiments, I/O interface 210 includes a keyboard, keypad, mouse, trackball, trackpad, or cursor direction keys for communicating information and commands to processor 202.

System architecture 200 also includes network interface 212 coupled to the processor 202. Network interface 212 allows computer architecture 200 to communicate with network 214, to which one or more other computer systems are connected. Network interface 212 includes wireless network interfaces such as BLUETOOTH, WIFI, WIMAX, GPRS, or WCDMA; or wired network interface such as ETHERNET, USB, or IEEE-1394. In some embodiments, a portion or all of the operations as described herein, and information are exchanged between different system architecture 200 via network 214.

In at least some embodiments, the apparatus is another device capable of processing logical functions in order to perform the operations herein. In at least some embodiments, the controller and the storage unit need not be entirely separate devices, but share circuitry or one or more computer-readable mediums in some embodiments. In at least some embodiments, the storage unit includes a hard drive storing both the computer-executable instructions and the data accessed by the controller, and the controller includes a combination of a central processing unit (CPU) and RAM, in which the computer-executable instructions are able to be copied in whole or in part for execution by the CPU during performance of the operations herein.

In at least some embodiments where the apparatus is a computer, a program that is installed in the computer is capable of causing the computer to function as or perform operations associated with apparatuses of the embodiments described herein. In at least some embodiments, such a program is executable by a processor to cause the computer to perform certain operations associated with some or all of the blocks of flowcharts and block diagrams described herein.

At least some embodiments are described with reference to flowcharts and block diagrams whose blocks represent (1) steps of processes in which operations are performed or (2) sections of a controller responsible for performing operations. In at least some embodiments, certain steps and sections are implemented by dedicated circuitry, programmable circuitry supplied with computer-readable instructions stored on computer-readable media, or processors supplied with computer-readable instructions stored on computer-readable media. In at least some embodiments, dedicated circuitry includes digital or analog hardware circuits and include integrated circuits (IC) or discrete circuits. In at least some embodiments, programmable circuitry includes reconfigurable hardware circuits including logical AND, OR, XOR, NAND, NOR, and other logical operations, flip-flops, registers, memory elements, etc., such as field-programmable gate arrays (FPGA) , programmable logic arrays (PLA) , etc.

In at least some embodiments, the computer readable storage medium includes a tangible device that is able to retain and store instructions for use by an instruction execution device. In some embodiments, the computer readable storage medium includes, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non- exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM) , a read-only memory (ROM) , an erasable programmable read-only memory (EPROM or Flash memory) , a static random access memory (SRAM) , a portable compact disc read-only memory (CD-ROM) , a digital versatile disk (DVD) , a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable) , or electrical signals transmitted through a wire.

In at least some embodiments, computer readable program instructions described herein are downloadable to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network or a wireless network. In at least some embodiments, the network includes copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers or edge servers. In at least some embodiments, a network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device.

In at least some embodiments, computer readable program instructions for carrying out operations described above are assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++ or the like, and conventional procedural programming languages, such as the C programming language or similar programming languages. In at least some embodiments, the computer readable program instructions are executed entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In at least some embodiments, in the latter scenario, the remote computer is connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN) , or the connection is made to an external computer (for example, through the Internet using an Internet Service Provider) . In at least some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA) , or programmable logic arrays (PLA) execute the computer readable program instructions by utilizing state information of the computer readable program instructions to individualize the electronic circuitry, in order to perform aspects of the subject disclosure.

While embodiments of the subject disclosure have been described, the technical scope of any subject matter claimed is not limited to the above described embodiments. Persons skilled in the art would understand that various alterations and improvements to the above-described embodiments are possible. Persons skilled in the art would also understand from the scope of the claims that the embodiments added with such alterations or improvements are included in the technical scope of the subject disclosure.

Figure 3 is a sequence diagram of a method 300 of rotating the stainer system in accordance with some embodiments.

A staining method comprising dynamically controlling an external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process 302. In some embodiments, the heating chamber being of low mass is capable of 304 being pressurized by external air, regulating the pressurization of the heating chamber using an adjustable air pressure control mechanism, 306 ensuring even heat distribution within a buffer pool using a capillary gap cover that protects a tissue during the HIER IHC staining process, 308 submerging a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling using a submersion system, 310 allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements using the heating chamber and associated components, and 312/314 rotating the heating chamber and the associated components so as to uniformly mix a reagent and a buffer.

The operations, procedures, steps, and stages of each process performed by an apparatus, system, program, and method shown in the claims, embodiments, or diagrams are able to be performed in any order as long as the order is not indicated by 'prior to, ' 'before, ' or the like and as long as the output from a previous process is not used in a later process. Even if the process flow is described using phrases such as 'first' or 'next' in the claims, embodiments, or diagrams, such a description does not necessarily mean that the processes must be performed in the described order.

Supplemental Note 1

A Legend 6G Stainer includes a heating chamber and associated components are capable of being rotated so as to uniformly mix a reagent and a buffer. The heating chamber is dynamically controlled external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, said heating chamber being of low mass and capable of being pressurized by external air. The Legend 6G Stainer further includes an adjustable air pressure control mechanism that regulates the pressurization of the heating chamber. The Legend 6G Stainer further includes a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool. The Legend 6G Stainer further includes a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling, the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements.

Supplemental Note 2

In some embodiments, the Legend 6G Stainer of Supplemental Note 1 wherein the heating chamber maintains a temperature range and a boing point between 100 and 130℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process.

Supplemental Note 3

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-2 further comprising a pressure control system that adjusts the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining.

Supplemental Note 4

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-3 wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.

Supplemental Note 5

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-4 wherein the capillary gap cover protects the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters.

Supplemental Note 6

A staining system includes a dynamically controlled external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, said heating chamber being of low mass and capable of being pressurized by external air. The staining system further includes an adjustable air pressure control mechanism configured to regulate the pressurization of the heating chamber. The staining system further includes a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool. The staining system further includes a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling, the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements. The Legend 6G Stainer further includes that the heating chamber and the associated components are capable of being rotated so as to uniformly mix a reagent and a buffer.

Supplemental Note 7

In some embodiments, the staining system of Supplemental Note 6 wherein the heating chamber maintains a temperature range and a boing point between 100 and 130℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process.

Supplemental Note 8

In some embodiments, the staining system of any of Supplemental Notes 6-7 further comprising a pressure control system configured to adjust the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining.

Supplemental Note 9

In some embodiments, the staining system of any of Supplemental Notes 6-8 wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.

Supplemental Note 10

In some embodiments, the staining system of any of Supplemental Notes 6-9 wherein the capillary gap cover protects the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters.

Supplemental Note 11

Supplemental Note 12

In some embodiments, the staining system of Supplemental Note 11 further comprising maintaining a temperature range and a boing point between 100 and 130℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process using the heating chamber.

Supplemental Note 13

In some embodiments, the staining method of any of Supplemental Notes 11-12 further comprising adjusting the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining using a pressure control system.

Supplemental Note 14

In some embodiments, the staining method of any of Supplemental Notes 11-13 wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.

Supplemental Note 15

In some embodiments, the staining method of any of Supplemental Notes 11-14 further comprising protecting the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters using the capillary gap cover.

Supplemental Note 16

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-4 wherein the heating chamber and the associated components are cable of being rotated back and forth between -8 and +8 degrees.

Supplemental Note 17

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-4 wherein the heating chamber and the associated components are cable of being rotated back and forth between -15 and +15 degrees.

Supplemental Note 18

In some embodiments, the Legend 6G Stainer of any of Supplemental Notes 1-4 wherein the heating chamber and the associated components are cable of being rotated back and forth between -30 and +30 degrees.

Claims

A Legend 6G Stainer comprising:

a dynamically controlled external air pressurized heating chamber and associated components capable of being rotated so as to uniformly mix a reagent and a buffer;

the heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process, the heating chamber being of low mass and capable of being pressurized by external air;

an adjustable air pressure control mechanism that regulates the pressurization of the heating chamber;

a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool; and

a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling, the heating chamber and the associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different pH and staining protocol requirements.
The Legend 6G Stainer of claim 1, wherein the heating chamber maintains a temperature range and a boiling point between 95℃ and 120℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process.
The Legend 6G Stainer of claim 2, further comprising a pressure control system that adjusts the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining.
The Legend 6G Stainer of claim 3, wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.
The Legend 6G Stainer of claim 4, wherein the capillary gap cover protects the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters.
A staining system comprising:

dynamically controlled external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process,

the heating chamber being of low mass and capable of being pressurized by external air;

an adjustable air pressure control mechanism configured to regulate the pressurization of the heating chamber;

a capillary gap cover that protects a tissue during the HIER IHC staining process to ensure even heat distribution within a buffer pool;

a submersion system that submerges a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling,

the heating chamber and associated components allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements, and

the heating chamber and the associated components are capable of being rotated so as to uniformly mix a reagent and a buffer.
The staining system of claim 6, wherein the heating chamber maintains a temperature range and a boing point between 100 and 130℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process.
The staining system of claim 7, further comprising a pressure control system configured to adjust the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining.
The staining system of claim 8, wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.
The staining system of claim 9, wherein the capillary gap cover protects the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters.
A staining method comprising:

dynamically controlling an external air pressurized heating chamber designed for a Heat Induced Epitope Retrieval (HIER) immunohistochemistry (IHC) staining process,

the heating chamber being of low mass and capable of being pressurized by external air;

regulating the pressurization of the heating chamber using an adjustable air pressure control mechanism;

ensuring even heat distribution within a buffer pool using a capillary gap cover that protects a tissue during the HIER IHC staining process;

submerging a slide within the buffer pool to eliminate formation of hot spots and micro nucleated boiling using a submersion system;

allowing users to adjust a pressure within a range of 8 to 12 psi, thereby providing control over a boiling point of the HIER IHC staining process for different tissue types and staining protocol requirements using the heating chamber and associated components; and

rotating the heating chamber and the associated components so as to uniformly mix a reagent and a buffer.
The staining method of claim 11, further comprising:

Maintaining a temperature range and a boing point between 100 95 and 130℃, reducing the risk of tissue damage and enabling precise control during the HIER IHC staining process using the heating chamber.
The staining method of claim 12, further comprising:

Adjusting the air pressure in real-time to prevent the formation of bubbles, ensuring effective antigen retrieval and staining using a pressure control system.
The staining method of claim 13, wherein the heating chamber comprises a size and liquid volume that eliminates formation of bubbles and allows for dynamic control of the HIER IHC staining process.
The staining method of claim 14, further comprising:

Protecting the slide from uneven heating and other irregularities that lead to formation of bubbles, preventing staining issues, and breaking off of cell clusters using the capillary gap cover.
The Legend 6G Stainer of claim 1, wherein the heating chamber and the associated components are cable of being rotated back and forth between -8 and +8 degrees.
The Legend 6G Stainer of claim 1, wherein the heating chamber and the associated components are cable of being rotated back and forth between -15 and +15 degrees.
The Legend 6G Stainer of claim 1, wherein the heating chamber and the associated components are cable of being rotated back and forth between -30 and +30 degrees.