A tuberculosis infection T cell immune spot test analyzer and analysis method

Abstract

This application discloses an analyzer and detection method for a T-cell immunospot assay for tuberculosis infection. The analyzer includes: a plate support structure for holding a completed test plate, and the plate support structure is provided with a position-limiting component for fixing the test plate; an imaging device moving component, including a drive structure arranged in multiple directions for driving the imaging device to move relative to the plate support structure in the front-back, left-right, and up-down directions; an image analysis module for receiving image data acquired by the imaging device, identifying and counting the reaction precipitates in the test wells based on the image data, and generating analysis results through a preset algorithm; and a plate configuration module for setting the test plate layout method and sample number information before analysis, the layout method including at least one layout pattern containing multiple different test well positions and control well positions.

Description

Technical Field

[0001] This application relates to the field of medical testing technology, and in particular to a tuberculosis infection T-cell immunospot assay analyzer and detection method. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis and remains a significant public health problem worldwide. Early and accurate diagnosis of TB infection is crucial for controlling its spread and ensuring timely treatment. T-cell immunospot assays (IGRAs), as an emerging immunological detection method, are widely used in the auxiliary diagnosis of TB infection. This method detects interferon-gamma (IFN-γ) spots released by peripheral blood mononuclear cells, reflecting the body's immune response to Mycobacterium tuberculosis-specific antigens, thereby determining the infection status.

[0003] Existing methods and equipment for T-cell immunospot assays for tuberculosis infection have shortcomings in terms of detection efficiency, automation, and accuracy of result interpretation, making it difficult to meet the clinical demand for rapid and accurate detection. Therefore, developing a highly efficient, automated, and accurate analyzer and detection method for T-cell immunospot assays for tuberculosis infection has significant clinical application value and broad market prospects. Summary of the Invention

[0004] To address the aforementioned technical issues, this application provides an instrument and method for analyzing the T-cell immunospot assay for tuberculosis infection.

[0005] The technical solution provided in this application is described below:

[0006] This application provides a tuberculosis infection T-cell immunospot assay analyzer, comprising: A plate support structure for placing a test plate that has completed the reaction, the plate support structure having a set color, and the plate support structure being provided with a position limiting component for fixing the test plate; An imaging device moving assembly includes a drive structure configured in multiple directions for driving the imaging device to move relative to the plate-supporting structure in the front-back, left-right, and up-down directions, wherein: The first driving structure is used to drive the imaging device to move in a first direction; The second driving structure is mounted on the first driving structure and is used to drive the imaging device to move in a second direction; The telescopic structure is used to drive the imaging device to move in a third direction; The image analysis module is used to receive image data collected by the imaging device, identify and count the amount of reaction precipitates in the test wells based on the image data, and generate analysis results through a preset algorithm. The plate configuration module is used to set the detection plate layout method and sample number information of the test plate before analysis. The plate layout method includes at least one plate layout mode that includes multiple different detection well positions and control well positions.

[0007] Optionally, the plate-supporting structure is white.

[0008] Optionally, the position limiting component includes elastic limiting members disposed on both sides of the plate bearing structure for limiting and fixing the test plate.

[0009] Optionally, the first drive structure includes two parallel first screws, the two ends of which are rotatably connected to the body of the analyzer and connected to the first drive motor via a transmission method.

[0010] Optionally, the second drive structure includes a second screw and a second drive motor. The second screw is connected to the second drive motor in a transmission connection, and a second threaded slider is threadedly connected to the middle of the second screw. The telescopic structure is connected to the second threaded slider.

[0011] Optionally, the telescopic structure includes a telescopic connecting rod disposed below the second threaded slider, and the imaging device is mounted on the lower end of the telescopic connecting rod.

[0012] A second aspect of this application provides a method for analyzing a tuberculosis infection T-cell immunospot assay, the method being applied to a tuberculosis infection T-cell immunospot assay analyzer as described in any one of claims 1 to 6, the method comprising: Add an appropriate amount of sterile saline or cell culture medium to the sample, mix well, and then slowly add it to a sample tube containing lymphocyte separation solution. Peripheral blood mononuclear cells were separated by density gradient centrifugation or extracted by magnetic bead method; The obtained mononuclear cell suspension was counted using a cell counter or cell counting chamber, and the mononuclear cell suspension was adjusted to a cell concentration of 250,000 cells / 100µl using cell culture medium. Take out the 96-well ELISA plate, add cell culture medium to the negative control wells, add Mycobacterium tuberculosis specific antigen to the antigen detection wells, and add positive control material to the positive control wells; Add mononuclear cell suspension to the corresponding well, cover the culture plate and incubate at 37°C, 5% CO2 for 16-20 hours; After culture, wash the plate to remove cells and non-specific conjugates, and add enzyme-labeled secondary antibody working solution for incubation; After incubation, wash the plate again, add the colorimetric solution, and incubate at room temperature in the dark. After color development, wash the plate to terminate the reaction, dry it, and place it in the placement area of ​​a 96-well plate.

[0013] According to the sample addition order, select the plate layout mode on the plate layout interface. The plate layout modes include: First plate layout: negative control well, antigen detection well, positive control well; Second plate layout: Negative control well, Antigen A detection well, Antigen B detection well, Positive control well After the stencil is completed, edit the sample number; The first driving structure is activated to drive the imaging device to move along the first direction; The second drive structure is activated to drive the imaging device to move along the second direction; The telescopic structure is activated, driving the imaging device to move along a third direction and position it above the enzyme label well on the corresponding test plate; Images of insoluble spots in the corresponding enzyme-labeled wells are acquired and transmitted to the image analysis module for processing.

[0014] Optionally, transmitting the insoluble spot image to the image analysis module for processing includes: The image analysis module counts the spots in the image based on an AI model; Set spot count thresholds for negative control wells, antigen detection wells or antigen A detection wells, antigen B detection wells, and positive control wells respectively; The test results are determined based on the plate pattern and the rule for comparing the number of spots, and the test results include at least "positive", "negative", and "uncertain".

[0015] Optionally, the first direction is the longitudinal movement direction along the test plate, the second direction is the transverse movement direction along the test plate, and the third direction is the vertical direction perpendicular to the test plate.

[0016] Optionally, peripheral blood mononuclear cells can be separated by density gradient centrifugation, including: The sample is slowly added to a sample tube containing lymphocyte separation fluid, and centrifuged at a specific speed and time to separate and collect the mononuclear cell layer.

[0017] As can be seen from the above technical solutions, this application has the following beneficial effects: This invention achieves rapid fixation and precise positioning of the test plate by setting up a plate-bearing structure with contrasting colors and a position limiting component, simplifying the plate-mounting operation process. Through the multi-drive structure linkage control of the imaging device's moving component along the three-dimensional direction, it can realize automated scanning and positioning of each well on the test plate, eliminating the need for manual plate movement and improving detection efficiency and laboratory throughput. Combined with the imaging device and image analysis module, it can capture reaction precipitation spots and accurately identify and count them based on a preset algorithm, significantly reducing human error and improving the repeatability and reliability of results. It enhances the system's adaptability to different experimental needs and can seamlessly interface with a Laboratory Information Management System (LIS) to achieve automatic result output and data management, greatly facilitating clinical applications and daily laboratory operations. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart illustrating an embodiment of the method provided in this application; Figure 2 This is a schematic diagram of a 96-well microplate. Figure 3 This is a schematic diagram of the interface for displaying spots in the enzyme-labeled wells; Figure 4 A schematic diagram of the plate layout interface for a 96-well microplate. Figure 5 This is a schematic diagram of the sample numbering interface; Figure 6 This is a schematic diagram of the results display interface. Figure 7 This is a schematic diagram of an embodiment of the tuberculosis infection T-cell immunospot assay analyzer provided in this application. Detailed Implementation

[0019] In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal" and other terms indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only used to describe the relative positional relationship between the components or parts and do not specifically limit the specific installation orientation of each component or part.

[0020] Furthermore, in addition to indicating location or positional relationship, some of the aforementioned terms may also have other meanings. For example, the term "above" may also be used in some cases to indicate a certain dependency or connection relationship. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0021] Furthermore, the terms "installation," "setup," "equipped with," "connection," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral structure; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0022] Furthermore, the structures, proportions, sizes, etc., drawn in the accompanying drawings of this application are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which this application can be implemented. Therefore, they have no substantial technical significance. Any modification to the structure, change in the proportional relationship, or adjustment of the size, without affecting the effects and purposes that this application can produce, should still fall within the scope of the technical content disclosed in this application.

[0023] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] See Figures 1 to 7 This application first provides an embodiment of a tuberculosis infection T-cell immunospot assay analyzer, which includes: The plate support structure 01 is used to place the test plate that has completed the reaction. The plate support structure has a set color and is provided with a position limiting component for fixing the test plate. The imaging device moving assembly 02 includes a driving structure arranged in multiple directions for driving the imaging device 03 to move relative to the plate bearing structure in the front-back, left-right, and up-down directions, wherein: The first driving structure 021 is used to drive the imaging device 03 to move in a first direction; The second driving structure 022 is mounted on the first driving structure and is used to drive the imaging device 03 to move in the second direction; The telescopic structure 023 is used to drive the imaging device to move in a third direction; The image analysis module is used to receive image data collected by the imaging device, identify and count the amount of reaction precipitates in the test wells based on the image data, and generate analysis results through a preset algorithm. The plate configuration module is used to set the detection plate layout method and sample number information of the test plate before analysis. The plate layout method includes at least one plate layout mode that includes multiple different detection well positions and control well positions.

[0025] The tuberculosis infection T-cell immune spot assay analyzer provided in this embodiment mainly includes a plate support structure, an imaging device moving component, an image analysis module, and a plate configuration module.

[0026] Plate-supporting structure: The plate support structure is used to hold the completed 96-well ELISA plate. This support structure is designed with a prominent color to facilitate quick identification and accurate placement of the plate by the operator.

[0027] To ensure the test plate remains stable and fixed during analysis, a position limiting component is provided on the plate's supporting structure. This position limiting component has a reasonable structure that matches the dimensions of the test plate, preventing the test plate from sliding or shifting during imaging scanning, thereby ensuring the accuracy and repeatability of the detection.

[0028] Imaging device moving components: The imaging device moving component includes a drive structure configured in multiple directions, which can drive the imaging device to move precisely relative to the plate support structure in three directions: front-back, left-right, and up-down, so as to realize automatic scanning of each hole in the test plate.

[0029] The first driving structure is used to drive the imaging device to move along a first direction (e.g., the front-to-back direction).

[0030] In one specific embodiment, the first driving structure may include a first driving motor 0211, a first screw 0213 and a first threaded slider 0212. The first driving motor drives the first screw to rotate, causing the first threaded slider to move back and forth along the screw, thereby driving the imaging device to achieve positioning movement.

[0031] The second drive structure is mounted on the first drive structure and is used to drive the imaging device to move along a second direction (e.g., left-right direction).

[0032] In one specific embodiment, the second driving structure includes a second driving motor, a second screw, and a second threaded slider. The second driving motor drives the second screw to rotate, causing the second threaded slider to move left and right, thereby realizing the lateral displacement of the imaging device.

[0033] The telescopic structure is used to drive the imaging device to move along a third direction (vertical direction). This structure can use a motor in conjunction with a mechanical telescopic rod to allow the imaging device to be adjusted vertically, ensuring that the imaging head can accurately focus on different depth positions of the test plate.

[0034] Through the coordinated operation of the aforementioned multi-directional driving structure, the imaging device can be freely positioned in three-dimensional space, ensuring accurate imaging of the reaction precipitate in each well of the ELISA plate.

[0035] Image analysis module The image analysis module is equipped with a high-performance processor and dedicated algorithms, enabling it to receive image data acquired by the imaging device and automatically identify and count the reaction precipitates within the test wells. Based on machine vision and artificial intelligence technologies, the algorithm analyzes the morphological features, size, and distribution of the spot images to automatically count the number of insoluble spots and generate the final analysis results according to pre-set judgment rules. This module supports real-time data processing and feedback, greatly improving the accuracy and efficiency of detection.

[0036] PCB configuration module The plate configuration module is used to set the test plate layout and sample numbering before analysis. This module supports at least two plate layout modes: The first plate layout includes negative control wells, antigen detection wells, and positive control wells, and is suitable for immunospot assays that detect single antigens.

[0037] The second plate layout includes negative control wells, antigen A detection wells, antigen B detection wells, and positive control wells, which are suitable for multi-antigen detection needs.

[0038] Operators can select the appropriate plate layout mode according to the specific testing task and edit the sample number information to ensure accurate archiving and management of test data.

[0039] In an optional embodiment, the plate-supporting structure is white.

[0040] In an optional embodiment, the plate-supporting structure is white. The technical advantages are: a white background has uniform high diffuse reflectance, which significantly improves the contrast between the immunospot colorimetric signal and the background, reduces imaging noise, and improves detection sensitivity, accuracy, and repeatability of results across multiple batches; it also clearly highlights the well outlines, numbers, and sample loading marks, reducing plate placement and loading errors and facilitating rapid verification of preparation quality before testing; it is also highly compatible with mainstream optical imaging system parameters, simplifying the calibration process, and easily detects contaminants for easy cleaning and maintenance; the accompanying medical-grade white polymer material has stable chemical properties, avoiding false-positive interference caused by non-specific colorimetry.

[0041] In an optional embodiment, the position limiting component includes elastic limiting members disposed on both sides of the plate bearing structure for limiting and fixing the test plate.

[0042] In one optional embodiment of this application, the position limiting component on the plate bearing structure includes elastic limiting members disposed on both sides of the plate bearing structure. The elastic limiting members can be made of elastic material, have a certain degree of flexibility and resilience, and can apply a moderate clamping force to both sides of the test plate placed on the bearing structure, thereby achieving the limiting and fixing of the test plate.

[0043] This elastic limiting component not only ensures that the test plate will not move or loosen during the imaging scanning process, but also makes it convenient for operators to quickly place and remove the test plate, improving the convenience of operation and the stability of the test.

[0044] The use of elastic limiting components enhances the adaptability and safety of the entire analyzer plate's load-bearing structure, making it suitable for test plates of different specifications and batches, and ensuring repeatability and accuracy during the imaging process.

[0045] In an optional embodiment, the first drive structure includes two parallel first screws, the two ends of which are rotatably connected to the body of the analyzer and connected to the first drive motor via a transmission method.

[0046] In an optional embodiment of this application, the first drive structure includes two parallel first screws. The two ends of each first screw are rotatably connected to the analyzer's body structure via rotating connectors, ensuring that the first screws can rotate smoothly while maintaining their spatial position.

[0047] The first screw is connected to the first drive motor through a transmission method such as gears, couplings or synchronous belts. When the drive motor rotates, it drives the two first screws to rotate synchronously, thereby realizing the movement of the first threaded slider connected to the first screw back and forth along the axial direction of the first screw.

[0048] This structural design ensures precise movement of the imaging device in the first direction, exhibiting good stability and repeatability, effectively improving the imaging positioning accuracy and detection efficiency of the analyzer.

[0049] In an optional embodiment, the second drive structure includes a second screw and a second drive motor. The second screw is connected to the second drive motor in a transmission manner, and a second threaded slider is threadedly connected to the middle of the second screw. The telescopic structure is connected to the second threaded slider.

[0050] In one optional embodiment of this application, the second drive structure includes a second screw and a second drive motor that is drively connected thereto. A second threaded slider is provided at the middle of the second screw and is threaded thereto.

[0051] The telescopic structure is installed and connected to the second threaded slider. The rotation of the second screw drives the second threaded slider to move along the axial direction of the second screw, thereby realizing the relative movement of the telescopic structure and the imaging device connected to it in the second direction.

[0052] This structural design enables the imaging device to achieve precise positioning in three directions: front-back, left-right, and up-down, improving the analyzer's automatic scanning capability and image acquisition efficiency for each hole on the test plate.

[0053] In an optional embodiment, the telescopic structure includes a telescopic connecting rod disposed below the second threaded slider, and the imaging device is mounted on the lower end of the telescopic connecting rod.

[0054] In an optional embodiment of this application, the telescopic structure includes a telescopic connecting rod disposed below the second threaded slider. The imaging device is fixedly mounted on the lower end of the telescopic connecting rod.

[0055] By driving the second threaded slider to move along the axial direction of the second screw, the telescopic connecting rod is driven to achieve vertical extension and retraction, thereby driving the imaging device to accurately position itself relative to the plate bearing structure in the third direction.

[0056] This design not only ensures stable support for the imaging device, but also allows the imaging device to flexibly adjust its height to adapt to the testing needs of different test plates, thereby improving the accuracy and reliability of the testing.

[0057] The following describes an embodiment of the tuberculosis infection T-cell immunospot assay analysis method provided in this application.

[0058] See Figure 1 Embodiments of the method provided in this application include: S101. Add an appropriate amount of sterile saline or cell culture medium to the sample, mix well, and then slowly add it to a sample tube containing lymphocyte separation solution. Remove the sample to be tested, add an appropriate amount of sterile saline or cell culture medium, and gently mix to ensure uniform cell suspension. Then, slowly add the mixed sample to a sample tube pre-filled with lymphocyte separation solution to prevent cell breakage or precipitation and ensure sample integrity and homogeneity.

[0059] S102. Peripheral blood mononuclear cells are separated by density gradient centrifugation or extracted by magnetic bead method; The sample tubes are placed in a centrifuge, and peripheral blood mononuclear cells (PBMCs) are separated using density gradient centrifugation based on differences in cell density. Alternatively, a magnetic bead method can be used, where target cells are captured and separated by specifically binding magnetic beads, improving cell purity. After extraction, the PBMC cell suspension is collected to ensure that cell viability and quantity meet the requirements for subsequent testing.

[0060] S103. Count the obtained mononuclear cell suspension using a cell counter or cell counting chamber, and adjust the mononuclear cell suspension to a cell concentration of 250,000 cells / 100µl using cell culture medium. The obtained PBMC suspension was counted using a cell counter or cell counting chamber to accurately calculate the cell concentration. The cell suspension was adjusted to 250,000 cells / 100µl using cell culture medium to ensure a consistent number of cells added to each well, thereby ensuring the reliability and comparability of the detection results.

[0061] S104. Take out the 96-well ELISA plate, add cell culture medium to the negative control wells, add Mycobacterium tuberculosis specific antigen to the antigen detection wells, and add positive control material to the positive control wells. Take out the 96-well ELISA plate and add reagents in sequence according to the preset experimental protocol: add cell culture medium to the negative control wells as a background control for the experiment; add Mycobacterium tuberculosis-specific antigen to the antigen detection wells to detect whether there is a tuberculosis infection T cell response in the sample; add positive control material to the positive control wells to verify the effectiveness of the experimental procedure.

[0062] S105. Add mononuclear cell suspension to the corresponding well, cover the culture plate and incubate at 37°C and 5% CO2 for 16-20 hours. Add the adjusted concentration of PBMC cell suspension to each corresponding well, cover the culture plate to prevent contamination, and place it in an incubator at 37°C and 5% CO2 for 16 to 20 hours to allow the cells to fully react and form specific spots.

[0063] S106. After the culture is completed, wash the plate to remove cells and non-specific conjugates, and add enzyme-labeled secondary antibody working solution for incubation. After culturing, remove the culture plate and use a plate washer or manual washing to remove unbound cells and non-specific binders from the wells. Add enzyme-labeled secondary antibody working solution and incubate for the specified time to achieve specific binding, ensuring the sensitivity and specificity of subsequent colorimetric reactions.

[0064] S107. After incubation, wash the plate again, add the color development solution and incubate at room temperature in the dark. After color development, wash the plate to stop the reaction, dry it and place it in the placement area of ​​a 96-well plate. After incubation, the plate was washed again to remove free secondary antibody, and the chromogenic solution was added. The plate was then incubated at room temperature in the dark. After the colorimetric reaction was complete, the plate was washed again to terminate the reaction. The plate was dried and then placed in the dedicated 96-well plate placement area, ready for imaging analysis.

[0065] S108. According to the sample addition order, select the plate layout mode on the plate layout interface. The plate layout modes include: First plate layout mode: negative control well, antigen detection well, positive control well; Second plate layout mode: negative control well, antigen A detection well, antigen B detection well, positive control well. According to the experimental sample addition sequence, select the appropriate plate layout mode in the analyzer's plate layout configuration interface to ensure that the system can accurately identify the function of each well position.

[0066] The first plate layout mode includes negative control wells, antigen detection wells, and positive control wells, suitable for simple experimental designs involving single antigen detection. The second plate layout mode includes negative control wells, antigen A detection wells, antigen B detection wells, and positive control wells, suitable for multi-antigen detection needs and supporting the analysis of complex samples.

[0067] S109. After the stencil is completed, edit the sample number; After selecting the plate layout mode, enter or edit the sample number information to ensure that each test data corresponds to the corresponding sample, which facilitates subsequent data management and traceability.

[0068] S110. Activate the first driving structure to drive the imaging device to move along the first direction; The first drive structure is activated, driving the imaging device to move precisely along a first direction (e.g., the front-to-back direction) to ensure that the scan covers each well of the experimental plate.

[0069] S111. Activate the second drive structure to drive the imaging device to move along the second direction; The second drive structure is activated, causing the imaging device to move along a second direction (e.g., left-right direction) to achieve positioning adjustment in a two-dimensional plane.

[0070] S112. Activate the telescopic structure to drive the imaging device to move along a third direction and position it above the enzyme label well on the corresponding test plate; Activate the telescopic structure to drive the imaging device to move along a third direction (up and down), adjust the height of the imaging device, and accurately position it above the target enzyme label well to ensure that the imaging device is aligned with the center of the well.

[0071] S113. Acquire images of insoluble spots in the corresponding enzyme-labeled wells and transmit the insoluble spot images to the image analysis module for processing.

[0072] The imaging device automatically adjusts the magnification and focal length to acquire images of insoluble spots generated in the corresponding enzyme-labeled wells, and transmits the image data to the image analysis module in real time. The image analysis module uses preset image recognition and statistical algorithms to automatically identify the number and distribution of spots, generating accurate analysis results for subsequent judgment and reporting.

[0073] In one optional embodiment, a method for analyzing a T-cell immunospot assay for tuberculosis infection is provided. This method combines an AI image recognition model to automatically count spots in multiple wells of an ELISA plate and interprets the results according to preset rules. The analysis method is applicable to different plate layouts and specifically includes spot counting rules and result interpretation rules.

[0074] First, the image acquisition module acquires the spot images of each well in the 96-well microplate, and the spot counting analysis algorithm identifies the number of spots in each well. After counting, the data is uploaded following the logic: Negative control well (N): When the number of spots is greater than 10, upload as if the number of spots is >10; Antigen detection well (T), Antigen A detection well (A), and Antigen B detection well (B): When the number of spots is greater than 200, upload as if the number of spots is >200; Positive control well (P): When the number of spots is greater than 20, upload the result as "number of spots > 20".

[0075] Based on this, and considering the different layout modes, the following result interpretation rules are set: First board layout mode: The first plate layout includes negative control wells (N), positive control wells (P), and antigen detection wells (T). The results interpretation rules are as follows: If the number of spots in the negative control well (N) is ≤ 10 and the number of spots in the positive control well (P) is ≥ 20, the data is considered a "normal result"; otherwise, the result is considered "uncertain". If the number of spots in the negative control well (N) is between 0 and 5, and the difference between the number of spots in the antigen detection well (T) and the number of spots in the negative control well (N) is ≥ 6, it is judged as "positive"; if the difference is less than 6, it is judged as "negative". If the number of spots in the negative control well (N) is between 6 and 10, and the number of spots in the antigen detection well (T) is ≥ twice the number of spots in the negative control well, it is considered "positive"; otherwise, it is considered "negative".

[0076] Second board layout mode: The second plate layout includes negative control wells (N), positive control wells (P), and antigen A detection wells (A) and antigen B detection wells (B). The result interpretation rules are as follows: If the number of spots in the negative control well (N) is ≤ 10 and the number of spots in the positive control well (P) is ≥ 20, the data is considered a "normal result"; otherwise, the result is considered "uncertain". If the number of spots in the negative control well (N) is between 0 and 5, and the difference between the number of spots in the antigen A detection well (A) or antigen B detection well (B) and the number of spots in the negative control well (N) is ≥ 6, it is judged as "positive"; if both are less than 6, it is judged as "negative". If the number of spots in the negative control well (N) is between 6 and 10, and the number of spots in the antigen A detection well (A) or antigen B detection well (B) is ≥ twice the number of spots in the negative control well (N), it is judged as "positive"; if both are less than twice, it is judged as "negative".

[0077] The method described in this embodiment can be deployed in an automated analysis system through program algorithms to achieve standardized uploading and intelligent interpretation of detection data, reduce human error, and improve detection efficiency and consistency.

Claims

1. A tuberculosis infection T-cell immunospot assay analyzer, characterized in that, include: A plate support structure for placing a test plate that has completed the reaction, the plate support structure having a set color, and the plate support structure being provided with a position limiting component for fixing the test plate; An imaging device moving assembly includes a drive structure configured in multiple directions for driving the imaging device to move relative to the plate-supporting structure in the front-back, left-right, and up-down directions, wherein: The first driving structure is used to drive the imaging device to move in a first direction; The second driving structure is mounted on the first driving structure and is used to drive the imaging device to move in a second direction; The telescopic structure is used to drive the imaging device to move in a third direction; The image analysis module is used to receive image data collected by the imaging device, identify and count the amount of reaction precipitates in the test wells based on the image data, and generate analysis results through a preset algorithm. The plate configuration module is used to set the detection plate layout method and sample number information of the test plate before analysis. The plate layout method includes at least one plate layout mode that includes multiple different detection well positions and control well positions.

2. The tuberculosis infection T-cell immunospot assay analyzer according to claim 1, characterized in that, The position limiting component includes elastic limiting members disposed on both sides of the plate bearing structure for limiting and fixing the test plate; the first driving structure includes two parallel first screws, the two ends of which are rotatably connected to the body of the analyzer and connected to the first driving motor through a transmission method.

3. The tuberculosis infection T-cell immunospot assay analyzer according to claim 1, characterized in that, The second driving structure includes a second screw and a second driving motor. The second screw is connected to the second driving motor in a transmission manner, and a second threaded slider is threadedly connected to the middle of the second screw. The telescopic structure is connected to the second threaded slider. The telescopic structure includes a telescopic connecting rod disposed below the second threaded slider, and the imaging device is installed at the lower end of the telescopic connecting rod.

4. A method for analyzing T-cell immunospot assays for tuberculosis infection, characterized in that, The method is applied to the tuberculosis infection T-cell immunospot assay analyzer as described in any one of claims 1 to 3, and the method comprises: Add an appropriate amount of sterile saline or cell culture medium to the sample, mix well, and then slowly add it to a sample tube containing lymphocyte separation solution. Peripheral blood mononuclear cells were separated by density gradient centrifugation or extracted by magnetic bead method; The obtained mononuclear cell suspension was counted using a cell counter or cell counting chamber, and the mononuclear cell suspension was adjusted to a cell concentration of 250,000 cells / 100µl using cell culture medium. Take out the 96-well ELISA plate, add cell culture medium to the negative control wells, add Mycobacterium tuberculosis specific antigen to the antigen detection wells, and add positive control material to the positive control wells; Add mononuclear cell suspension to the corresponding well, cover the culture plate and incubate at 37°C, 5% CO2 for 16-20 hours; After culture, wash the plate to remove cells and non-specific conjugates, and add enzyme-labeled secondary antibody working solution for incubation; After incubation, wash the plate again, add the color development solution and incubate at room temperature in the dark. After color development, wash the plate to stop the reaction, dry it and place it in the placement area of ​​a 96-well plate. According to the sample addition order, select the plate layout mode on the plate layout interface. The plate layout modes include: First plate layout: negative control well, antigen detection well, positive control well; Second plate layout: Negative control well, Antigen A detection well, Antigen B detection well, Positive control well After the stencil is completed, edit the sample number; The first driving structure is activated to drive the imaging device to move along the first direction; The second drive structure is activated to drive the imaging device to move along the second direction; The telescopic structure is activated, driving the imaging device to move along a third direction and position it above the enzyme label well on the corresponding test plate; Images of insoluble spots in the corresponding enzyme-labeled wells are acquired and transmitted to the image analysis module for processing.

5. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, The step of transmitting the insoluble spot image to the image analysis module for processing includes: The image analysis module counts the spots in the image based on an AI model; Set spot count thresholds for negative control wells, antigen detection wells or antigen A detection wells, antigen B detection wells, and positive control wells respectively; The test results are determined based on the plate pattern and the rule for comparing the number of spots, and the test results include at least "positive", "negative" and "uncertain".

6. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, The first direction is the longitudinal movement direction along the test plate, the second direction is the transverse movement direction along the test plate, and the third direction is the vertical direction perpendicular to the test plate.

7. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, Peripheral blood mononuclear cells were isolated by density gradient centrifugation, including: The sample is slowly added to a sample tube containing lymphocyte separation solution, and peripheral blood mononuclear cells are separated using specific centrifugation conditions of speed and time.

8. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, The detection results are determined based on the plate pattern and the rule for comparing the number of spots, including: Negative control well: When the number of spots is greater than 10, upload as if the number of spots is >10; Antigen detection wells, antigen A detection wells, and antigen B detection wells: When the number of spots is greater than 200, upload as if the number of spots is >200. Positive control well: When the number of spots is greater than 20, upload as if the number of spots is >20.

9. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, In the first plate layout mode, a "normal result" is defined as a negative control well with ≤10 spots and a positive control well with ≥20 spots; otherwise, it is defined as "uncertain". If the number of spots in the negative control well is 0 to 5, and the number of spots in the antigen detection well minus the number of spots in the negative control well is ≥ 6, it is judged as "positive"; if it is less than 6, it is judged as "negative". If the number of spots in the negative control well is 6 to 10, and the number of spots in the antigen detection well is ≥ twice the number of spots in the negative control well, it is judged as "positive"; otherwise, it is "negative".

10. The method for analyzing tuberculosis infection T-cell immunospot assay according to claim 4, characterized in that, A result is considered "normal" if the number of spots in the negative control well is ≤10 and the number of spots in the positive control well is ≥20; otherwise, it is considered "uncertain". If the number of spots in the negative control well is 0 to 5, and the number of spots in the antigen A detection well minus the number of spots in the negative control well is ≥ 6, it is judged as "positive"; if both are less than 6, it is judged as "negative". If the number of spots in the negative control well is 6 to 10, and the number of spots in the antigen A detection well or the antigen B detection well is ≥ twice the number of spots in the negative control well, it is judged as "positive"; if both are less than twice, it is judged as "negative".

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