Indirect immunofluorescence detection method and system for single person

Abstract

The invention relates to a single-person indirect immunofluorescence detection method and a single-person indirect immunofluorescence detection system. A single-person reaction container is used as a fixing and detection platform for indirect immunofluorescence detection; an independent sample detection process is realized by adopting automatic liquid treatment and optical imaging; image features are automatically processed, and signals are quantized and output in a classified mode. Indirect immunofluorescence detection is directly optimized and designed from the aspect of a single-person reaction container, the problem of batch processing delay is solved, and indirect immunofluorescence detection is directly carried out on a single sample; the problem of limited degree of automation is solved, samples do not need to be transferred manually, optical imaging does not need to be set manually, and a full-automatic indirect immunofluorescence detection system is realized; the problems of sample confusion and low flexibility are solved, batch glass slides do not need to be repeatedly utilized, and emergency samples can be quickly inserted into a detection queue; the device solves the problem of low expandability, can be cascaded with a traditional analysis instrument, can be used in parallel connection with a plurality of single-thread detection devices, and is suitable for laboratories of various scales.

Description

Technical Field

[0001] This application relates to the field of automated analysis systems, and in particular to a method and system for single-sample indirect immunofluorescence detection. Background Technology

[0002] Indirect immunofluorescence (IIF) is a widely used technique for detecting specific antigens in biological samples such as cells or tissues using fluorescently labeled antibodies. It has been applied in the screening and diagnosis of autoimmune diseases, which is beneficial for early detection and prevention and treatment, and has a great impact on patients' lifespan and quality of life.

[0003] Traditional IIF methods rely on slides for batch processing, including manual sample coating, antibody incubation, cleaning, and fluorescence microscopy observation. This method is labor-intensive, error-prone, and inefficient in high-throughput applications.

[0004] With the rise of automated systems, some detection functions have been automated, but batch processing is still the main method, processing 3 to 100 samples or even more at a time, but it cannot flexibly process individual samples.

[0005] Traditional detection methods primarily rely on glass slides as carriers. Typically, cells or tissue sections are coated onto the slide, which is then cut into millimeter-sized slices or left uncut. One or more slices are then attached to the reaction wells of the slide, along with other supporting reagents, to form the detection reagent product. To fully utilize resources, this method requires batch processing, such as 3, 5, 10, or 50 samples, including sample coating, antibody incubation, washing, and fluorescence detection. This slide-based batch method is effective for antigen detection, but it usually requires manual or semi-automated operation and cannot achieve independent testing for single individuals.

[0006] For a slide with 10 reaction wells, if 10 samples are tested per slide, the reaction wells can be fully utilized. However, during use, a single test must reach 10 samples. If the number of samples tested is less than 10, it will result in waste of reagents. If 3 or 5 samples are tested per slide, and only 3 or 5 reaction wells are used, the remaining 7 or 5 wells will be idle and wasted, leading to material waste and increasing the cost per reagent.

[0007] Traditional detection techniques have the following problems:

[0008] 1. Batch processing delay. Existing methods process samples in fixed batches, resulting in a long time from the first sample to the completion of the batch, usually no less than 2 hours. This delays the results of urgent samples and affects time-sensitive diagnostics.

[0009] 2. Risks associated with manual operation. Steps involving manual handling of slides, such as sample coating, slide transfer, and cleaning, increase the risk of human error, contamination, and sample loss, especially in high-throughput laboratories where contamination issues are more severe.

[0010] 3. Sample confusion and low flexibility. Using multiple slides in batches can easily lead to labeling errors or confusion, making it impossible to process individual samples as needed and reducing workflow adaptability.

[0011] 4. Limited automation. Although some steps, such as cleaning, are automated, the overall system still relies on manual preparation of transfer samples and imaging setup. The system is complex and expensive, and therefore not widely adopted, especially in resource-constrained environments.

[0012] 5. Low scalability. The manual nature and batch processing hinder the scaling up of testing, making it difficult to meet the growing diagnostic needs and directly limiting the clinical applicability and efficiency of the method. Summary of the Invention

[0013] Therefore, it is necessary to provide a single-sample indirect immunofluorescence detection method and system.

[0014] One embodiment of this application is a single-sample indirect immunofluorescence detection method, which includes the following steps:

[0015] Single-use reaction containers were used as a fixation and detection platform for indirect immunofluorescence assays.

[0016] An automated liquid handling and optical imaging process is used to achieve independent sample detection.

[0017] It automatically processes image features, quantizes signals, and classifies patterns for output.

[0018] The aforementioned single-sample indirect immunofluorescence assay method introduces the concept of single-sample testing, directly optimizing the design of the single-sample reaction vessel. This addresses the issue of batch processing delays, eliminating the need for samples to wait for batch testing of other samples; instead, it directly performs indirect immunofluorescence assays on individual samples, thus reducing waiting time and improving detection efficiency. Furthermore, the shift from batch to single-sample design helps reduce product footprint. On the other hand, it overcomes the limitation of automation, eliminating the need for manual sample transfer and optical imaging setup, facilitating a fully automated indirect immunofluorescence assay system and improving clinical efficacy. It offers the advantages of a single-use reaction vessel, which improves both practicality and testing efficiency while avoiding the risks associated with manual operation. Furthermore, it addresses the issues of sample confusion and low flexibility. The single-use reaction vessel is applied throughout the entire testing process, eliminating the need for reusing traditional batch slides. Because it operates as an independent sample testing process, urgent samples can be quickly inserted into the testing queue, facilitating emergency diagnosis and further enhancing clinical practicality and flexibility. It also solves the problem of low scalability by eliminating manual and batch processes. It is compatible with traditional testing and analysis systems, can be cascaded with traditional analytical instruments, and allows for parallel use of multiple single-threaded tests. Therefore, it can be flexibly configured according to needs and is suitable for laboratories of various sizes.

[0019] As an example, in an automated liquid handling and optical imaging process for independent sample detection, indirect immunofluorescence treatment is performed on samples in a single-use reaction vessel. This indirect immunofluorescence treatment includes specific binding, fluorescent labeling, and signal amplification. Specific binding involves fixing the sample (e.g., tissue sections or cell smears), adding an unlabeled specific primary antibody, and incubating it to allow the primary antibody to specifically bind to the target molecule in the sample. Fluorescent labeling and signal amplification involve adding a fluorescein-labeled secondary antibody, which binds to the primary antibody that has already bound to the target. Since one primary antibody can bind to multiple secondary antibodies, signal amplification is ultimately achieved, allowing observation of the target molecule's location and expression using fluorescence microscopy.

[0020] In some embodiments, using a single-use reaction container as a fixation and detection platform for indirect immunofluorescence detection includes: using a single-use reaction container to contain a sample, and using the single-use reaction container containing the sample as a fixation and detection platform for indirect immunofluorescence detection, to add reagents and carry out a reaction.

[0021] In some embodiments, automated liquid handling and optical imaging are used to implement an independent sample detection process, including:

[0022] The single-use reaction vessel is transported to a fixed position, and a fixation reagent is added to the single-use reaction vessel containing the sample to achieve sample fixation.

[0023] The single-use reaction container is delivered to the reaction location, and reaction reagents are added to the single-use reaction container containing the sample to achieve antibody incubation and washing.

[0024] A single-use reaction vessel is transported to the detection location, and a fluorescence image is acquired from the bottom of the single-use reaction vessel.

[0025] In some embodiments, each single-dose reaction container is identified, and the single-dose reaction container is delivered to the fixed position, reaction position, and detection position in a continuous random sampling and sequential detection manner; or, the single-dose reaction container is delivered to the fixed position, reaction position, and detection position in a first-in-first-out combined with priority identification manner.

[0026] In some embodiments, the single-use reaction vessel is a single-use flat-bottomed reaction vessel to acquire fluorescence images from the bottom of the single-use reaction vessel.

[0027] In some embodiments, automated liquid handling and optical imaging are used to implement independent sample detection processes, including: independent timing and / or metrological processing for each individual process.

[0028] In some embodiments, image features are processed automatically, including using large artificial intelligence models to automatically identify and process image features obtained from optical imaging.

[0029] In some embodiments, quantization of the signal includes quantifying image features in a standardized manner, calculating fluorescence intensity, and providing digital grading results.

[0030] In some embodiments, the classification mode output includes standard classification based on nuclear homogeneity, nuclear granules, nucleolarity, and cytoplasmic type, combined with an artificial intelligence model for auxiliary interpretation, and output of a test report.

[0031] In some embodiments, a single-person indirect immunofluorescence detection system includes:

[0032] Single-use reaction containers serve as a fixation and detection platform for indirect immunofluorescence assays;

[0033] The indirect immunofluorescence processing unit employs automated liquid handling and optical imaging to achieve an independent sample detection process; and,

[0034] The analysis unit is used to automatically process image features, quantize signals, and classify pattern outputs. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application or the conventional technology, the drawings used in the description of the embodiments or the conventional technology will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic flowchart of an embodiment of the single-sample indirect immunofluorescence detection method described in this application. Detailed Implementation

[0037] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0038] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on the other component or there may be an intermediate component. When a component is considered to be "connected to" another component, it can be directly connected to the other component or there may be an intermediate component present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used in this application's specification are for illustrative purposes only and do not represent the only possible implementation.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0040] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature and the second feature are in indirect contact through an intermediate medium. Furthermore, "above," "over," and "on top" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0041] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and or" as used in this application includes any and all combinations of one or more of the associated listed items.

[0042] This application discloses a single-sample indirect immunofluorescence detection method and system, which includes some or all of the technical features of the following embodiments; for example, the single-sample indirect immunofluorescence detection method includes some or all of the following steps. In one embodiment of this application, a single-sample indirect immunofluorescence detection method is as follows: Figure 1 As shown, the method includes the following steps: using a single-use reaction container as the fixation and detection platform for indirect immunofluorescence detection; employing automated liquid handling and optical imaging to achieve an independent sample detection process; and automatically processing image features, quantifying signals, and outputting classification patterns. This single-use indirect immunofluorescence detection method introduces the concept of single-use detection, directly optimizing the design of the indirect immunofluorescence detection from the perspective of the single-use reaction container. On the one hand, it solves the problem of batch processing delays, as samples do not need to wait for other samples to undergo batch testing, but can directly undergo indirect immunofluorescence detection on individual samples. This reduces the waiting time for individual samples and improves detection efficiency. Moreover, the shift from batch to single-use detection design helps reduce the product's footprint. On the other hand, it solves the problem of limited automation, eliminating the need for manual sample transfer and manual optical imaging setup, facilitating the realization of a fully automated indirect immunofluorescence detection system, thereby improving clinical application efficiency. It offers the advantages of a single-use reaction vessel, which improves both practicality and testing efficiency while avoiding the risks associated with manual operation. Furthermore, it addresses the issues of sample confusion and low flexibility. The single-use reaction vessel is applied throughout the entire testing process, eliminating the need for reusing traditional batch slides. Because it operates as an independent sample testing process, urgent samples can be quickly inserted into the testing queue, facilitating emergency diagnosis and further enhancing clinical practicality and flexibility. It also solves the problem of low scalability by eliminating manual and batch processes. It is compatible with traditional testing and analysis systems, can be cascaded with traditional analytical instruments, and allows for parallel use of multiple single-threaded tests. Therefore, it can be flexibly configured according to needs and is suitable for laboratories of various sizes.

[0043] The study found that the biggest limitations of traditional techniques in clinical applicability and efficiency lie in batch processing delays and insufficient automation. In various embodiments, single-use reaction containers are used as the fixation and detection platform for indirect immunofluorescence detection. In some embodiments, using a single-use reaction container as the fixation and detection platform for indirect immunofluorescence detection includes: using a single-use reaction container to hold the sample, and using the single-use reaction container containing the sample as the fixation and detection platform for adding reagents and initiating the reaction. That is, for subsequent steps or processes, the single-use reaction container is used throughout the entire indirect immunofluorescence detection process, from sample addition to detection completion. The single-use reaction container corresponds one-to-one with the sample, and the sample remains on the same single-use reaction container throughout, eliminating the need to transfer the sample to a slide as in traditional techniques, thus achieving single-use reaction container integration. As an example, the single-use reaction container uses a flat-bottomed, high-transmittance reaction container, completing sample fixation, antibody incubation, washing, and fluorescence detection in a single container, avoiding slide transfer and batch processing delays. As an example, the single-use reaction container is a single-use flat-bottomed reaction container, and it is moved to the corresponding operating position during the execution of the single-use indirect immunofluorescence detection method.

[0044] This design effectively addresses the core issue of batch processing delays in traditional technologies. By using a single-use reaction container as the fixation and detection platform for indirect immunofluorescence detection, the sample remains in the same container from addition to the end of the test. It eliminates the need to transfer samples to traditional slides or wait for other samples to form a batch, significantly reducing the waiting time for individual samples and greatly improving detection efficiency. Furthermore, the single-use design eliminates the need for large support structures required for batch processing, reducing the space occupied by the detection equipment and adapting to the site requirements of laboratories of different sizes. On the other hand, it effectively overcomes the drawbacks of insufficient automation. The single-use reaction container is used throughout the entire detection process, and combined with automated liquid handling, it enables automated operations such as reagent addition and reaction control, eliminating the need for manual sample transfer or process intervention. Especially when using a flat-bottomed, high-transmittance reaction container, the entire process of sample fixation, antibody incubation, cleaning, and fluorescence detection can be completed within a single container, completely avoiding the risks of sample contamination and operational errors caused by manual operation. This helps to build a fully automated indirect immunofluorescence detection system and improves the reliability of clinical applications. On the other hand, it addresses the issue of low flexibility in traditional batch testing. Each single-use reaction container corresponds to a single sample, eliminating the need to reuse slides. The same single-use reaction container is maintained for the same sample throughout the entire testing process, and urgent samples can be quickly inserted into the testing queue without waiting for the entire batch of samples to be processed. This allows for timely response to urgent diagnostic needs, further enhancing clinical applicability and testing flexibility. At the same time, the single-use reaction container can be moved to the corresponding operating position during the testing process, adapting to the workflow design of automated production lines. This lays the foundation for subsequent cascading with other automated equipment, improving the overall adaptability of the testing system.

[0045] In each embodiment, automated liquid handling and optical imaging are used to implement an independent sample detection process. As an example, in this process, indirect immunofluorescence treatment is performed on samples in a single-use reaction vessel. As an example, indirect immunofluorescence treatment includes specific binding and fluorescent labeling and signal amplification. Specific binding involves fixing the sample to be tested, such as a tissue section or cell smear, and adding an unlabeled specific primary antibody. After incubation, the primary antibody specifically binds to the target molecule in the sample. Fluorescent labeling and signal amplification involve adding a fluorescein-labeled secondary antibody, which binds to the primary antibody that has already bound to the target. Since one primary antibody can bind to multiple secondary antibodies, signal amplification is ultimately achieved, allowing observation of the target molecule's location and expression using fluorescence microscopy or similar methods.

[0046] This design, on the one hand, establishes an independent sample detection process through automated liquid handling and optical imaging. Indirect immunofluorescence processing is completed within a single-use reaction container, eliminating the need for manual intervention in liquid addition and imaging. This avoids problems such as reagent dosage errors and uneven incubation times inherent in traditional manual methods, ensuring consistency in processing conditions for each sample and improving the accuracy and repeatability of test results. Furthermore, the independent process eliminates the need to wait for batch samples, further shortening the testing cycle and enhancing clinical application efficiency. On the other hand, the step-by-step design of specific binding, fluorescent labeling, and signal amplification first uses an unlabeled specific primary antibody to precisely bind to the target molecule in the sample. Then, a fluorescein-labeled secondary antibody binds to the primary antibody, utilizing the binding characteristics of the primary antibody with multiple secondary antibodies to achieve signal amplification. This clearly reveals the location and expression status of low-expression target molecules, solving the problem of weak and difficult-to-identify low-abundance target signals in traditional detection methods and improving detection sensitivity. Moreover, the entire process is completed within a single-use reaction container, eliminating the need for sample transfer and avoiding the risk of target molecule loss or sample contamination during transfer.

[0047] For automated processing flow operations, in some embodiments, automated liquid handling and optical imaging are used to achieve independent sample testing processes, including: transporting a single-use reaction container to a fixed position, adding fixation reagents to the single-use reaction container containing the sample to achieve sample fixation; transporting the single-use reaction container to a reaction position, adding reaction reagents to the single-use reaction container containing the sample to achieve antibody incubation and washing; transporting the single-use reaction container to a detection position, and acquiring fluorescence images from the bottom of the single-use reaction container. As an example, the same set of automated robotic arms can be used to complete the above-mentioned transport of single-use reaction containers to the fixed position, reaction position, and detection position, or different automated robotic arms can be used in conjunction with transport tracks to transport single-use reaction containers to the fixed position, reaction position, and detection position. As an example, the single-use reaction containers are transported to the fixed position, reaction position, and detection position on the same plane, that is, during the transport of the single-use reaction containers, the height of the single-use reaction containers, i.e., the altitude or horizontal level, remains unchanged.

[0048] This design, on the one hand, establishes a standardized workflow through automated liquid handling and optical imaging. Single-use reaction containers are sequentially transported to fixed, reaction, and detection positions, respectively completing sample fixation, antibody incubation and cleaning, and fluorescence image acquisition. The entire process requires no manual intervention for sample transfer or reagent addition, completely resolving the problems of chaotic steps and uneven processing time in traditional manual operations. This ensures consistency in each sample testing process, significantly reduces human error and sample contamination risks, and significantly improves the reliability and repeatability of test results, further enhancing the stability of clinical applications. On the other hand, the flexible transport design combines practicality and adaptability: either a single set of automated robotic arms can complete the entire transport process, simplifying equipment structure, reducing component redundancy, and lowering equipment procurement and maintenance costs; or different automated robotic arms can be used in conjunction with transport tracks to achieve segmented transport, adapting to high-throughput testing needs and improving overall operational efficiency. Both transport options can be flexibly selected according to laboratory size and testing volume, enhancing compatibility with different application scenarios. On the other hand, using a single-person reaction container to transport it on the same plane keeps its height constant, avoiding spillage of samples or reagents or fluctuations in the liquid level due to height changes during transport. This ensures the stability of key steps such as sample fixation and incubation reaction, and prevents changes in liquid state from affecting the detection results. At the same time, the planar transport eliminates the need for complex vertical lifting mechanisms, simplifying mechanical design, reducing the probability of mechanical failure, and further ensuring the continuous and smooth operation of automated production lines.

[0049] In some embodiments, each single-dose reaction container is identified, and the single-dose reaction container is delivered to a fixed position, a reaction position, and a detection position using a continuous random sampling and sequential detection method; or, in some embodiments, each single-dose reaction container is identified, and the single-dose reaction container is delivered to a fixed position, a reaction position, and a detection position using a first-in-first-out combined with priority identification method.

[0050] This design, on the one hand, provides clear evidence for sample traceability by identifying each individual reaction container, avoiding sample confusion problems in traditional batch testing, ensuring accurate correspondence between test results and samples, improving the reliability of clinical diagnosis, and reducing the risk of misdiagnosis due to sample mismatch. On the other hand, the two delivery methods flexibly adapt to different testing needs: continuous random sampling and sequential testing can flexibly schedule samples according to the actual testing plan to meet diverse testing and sequencing requirements; the first-in-first-out (FIFO) combined with priority identification method can ensure orderly testing of routine samples while quickly responding to urgent sample needs, prioritizing the delivery of urgent samples to the corresponding locations, shortening the time required for emergency diagnosis, and adapting to testing and scheduling needs in different scenarios.

[0051] In some embodiments, the single-use reaction vessel is a single-use flat-bottomed reaction vessel to acquire fluorescence images from its bottom. Acquiring fluorescence images from the bottom of the single-use reaction vessel occupies less space above and does not affect automated liquid handling equipment, thus reducing the overall system's space requirements. Furthermore, for optical imaging, fewer adjustments are needed, maintaining optical path stability and significantly improving imaging efficiency. As an example, the single-use reaction vessel can be a cuvette or other flat-bottomed glass container holding or containing one sample; tubular glass containers, especially flat-bottomed ones, can also be used.

[0052] This design offers several advantages. First, it utilizes a single-use flat-bottomed reaction vessel as the carrier, acquiring fluorescence images from its bottom. This bottom-imaging method occupies minimal space above the vessel, avoiding spatial conflicts with automated liquid handling equipment. It eliminates the need for extra space to accommodate imaging components, effectively compressing the overall size of the single-use indirect immunofluorescence detection system. This facilitates deployment in space-constrained laboratory settings, improving equipment site adaptability. Second, the bottom-imaging design significantly reduces the need for optical imaging adjustments. It allows for stable fluorescence signal capture without frequent adjustments to lens angles or vessel orientation, maintaining long-term optical path stability and preventing imaging deviations caused by frequent position changes. This significantly reduces optical setup time, greatly improves imaging efficiency, and ensures a continuous and smooth detection process. Third, the single-use flat-bottomed reaction vessel can be a cuvette or other flat-bottomed glass container. These containers have high light transmittance, reducing fluorescence signal loss during transmission and ensuring image clarity. Furthermore, their standardized flat-bottom structure is compatible with various optical imaging modules, eliminating the need to adjust imaging parameters for different container shapes, further improving detection efficiency and accuracy.

[0053] In some embodiments, automated liquid handling and optical imaging are used to implement independent sample testing processes, including independent timing or measurement for each individual process. For example, for each single-use reaction vessel and the sample it contains, a separate process with a sequential or priority order is established. Each individual process undergoes independent continuous testing, including independent timing and measurement, and this continuous processing eliminates the need to wait for batches. Unlike traditional batch testing methods, each single-use reaction vessel corresponding to a sample can be uniquely identified by a QR code, preventing confusion or contamination when transferring to slides for multiple samples. For example, with independent timing for each sample, the incubation time is reduced to within 10 to 15 minutes, and the entire process is continuously processed for 45 minutes, supporting continuous random sampling and sequential testing, improving flexibility and throughput.

[0054] This design addresses the core pain point of process-bound processes in traditional batch testing at its root. By independently timing or timing and measuring each individual process, the testing steps for each sample are no longer limited by the progress of other samples, eliminating the need to wait for the entire batch to be processed simultaneously. This completely breaks free from the batch constraints of traditional batch testing. For example, by establishing a separate process for each individual reaction container, the amount of reagent added and the incubation time can be precisely controlled according to the characteristics of the sample. This avoids the problems of under-processing or over-processing of some samples caused by the averaging operation in traditional batch processing, significantly improving the accuracy and repeatability of test results. At the same time, it saves the time spent waiting for batch processing and significantly speeds up the testing of individual samples, providing support for rapid clinical diagnosis. On the other hand, by using identification such as barcodes or QR codes, each individual reaction container is given a unique identifier. Combined with the independent process design, this enables full traceability of the sample from start to finish of testing. Samples do not need to be transferred to multi-person slides, avoiding the risks of sample confusion and cross-contamination caused by operational errors during transfer. It also eliminates the tedious steps of repeated slide cleaning and labeling, further ensuring testing safety and process simplicity, and solving the problems of difficult sample traceability and high contamination risk in traditional batch testing. Furthermore, by optimizing the efficiency of key steps through independent timing, the incubation time is shortened to within 10 to 15 minutes, maintaining continuous processing for the entire process within 45 minutes, significantly reducing the total testing time. It also supports continuous random sampling and sequential testing, efficiently processing samples in the conventional order while flexibly inserting emergency samples without disrupting the original testing queue, balancing throughput and emergency response capabilities. This design can also flexibly adjust the operating scale according to the laboratory's testing volume, adapting to the dispersed sample testing needs of small laboratories and meeting the high-throughput needs of large laboratories through multi-unit parallel connection, further enhancing the scenario adaptability and clinical applicability of the single-person indirect immunofluorescence detection method.

[0055] Each embodiment employs automated image acquisition and analysis, i.e., automated processing of image features, quantification of signals, and classification pattern output. In some embodiments, automated image feature processing includes using a large-scale artificial intelligence model to automatically identify and process image features obtained from optical imaging. In some embodiments, signal quantification includes quantifying image features using a standardized processing method, calculating fluorescence intensity, and providing digital grading results. In some embodiments, classification pattern output includes standard classification based on homogeneous, granular, nucleolar, and cytoplasmic morphologies, supplemented by an artificial intelligence model for assisted interpretation, and outputting a test report. As an example, fluorescence images are acquired directly from the bottom of a container using an inverted microscope, integrating artificial intelligence for self-interpretation, automatic titer calculation, and karyotype discrimination, achieving fully automated result output without human intervention. In some embodiments, automated image feature processing, signal quantification, and classification pattern output specifically include: using a large-scale artificial intelligence model to automatically identify and process image features obtained from optical imaging; quantifying image features using a standardized processing method, calculating fluorescence intensity, and providing digital grading results; performing standard classification based on homogeneous, granular, nucleolar, and cytoplasmic morphologies, supplemented by an artificial intelligence model for assisted interpretation, and outputting a test report. The other embodiments follow the same pattern and will not be described in detail.

[0056] This design, on the one hand, replaces traditional manual interpretation with automated image acquisition and analysis, completely solving the problems of high subjectivity and low efficiency associated with manual operation. Employing a large-scale artificial intelligence model to automatically identify image features in optical imaging, it can accurately capture subtle differences in fluorescence signals, avoiding omissions or misjudgments that can occur with manual visual identification. Combined with standardized processing to quantify image features, calculate fluorescence intensity, and provide digital grading results, the test results are more objective and comparable, significantly improving accuracy and consistency. On the other hand, standard classification based on homogeneous, granular, nucleolar, and cytoplasmic morphologies, combined with the AI-assisted interpretation model, ensures that the classification results meet clinical standards, while rapidly outputting test reports, significantly shortening the time required for result analysis and report generation. In particular, by combining fluorescence images acquired from the bottom of the container using an inverted microscope, and integrating AI for self-interpretation, automatic titer calculation, and karyotype discrimination, it achieves fully automated result output without human intervention, further simplifying the testing process, reducing errors and risks associated with manual operation, and facilitating the full automation of single-sample indirect immunofluorescence detection methods. This significantly improves clinical testing efficiency, thereby meeting the clinical demand for rapid and accurate diagnosis.

[0057] Based on any embodiment of the single-sample indirect immunofluorescence detection method, as an example, the single-sample indirect immunofluorescence detection method further includes the step of: using dynamic resource scheduling to adjust the position of the single-sample reaction container in the sample detection queue to achieve priority processing of urgent samples, ensure efficient resource utilization and seamless report generation, be compatible with sequential and priority processing, and be fully integrated into an automated fluorescence detection and analysis system, thereby achieving efficient and independent sample processing.

[0058] This design addresses the pain point of prioritizing urgent samples in traditional batch testing by dynamically adjusting the position of single-sample reaction containers in the sample testing queue through resource scheduling. When an urgent sample enters the testing process, its queue order can be flexibly adjusted, eliminating the need to wait for regular samples to complete testing. This allows for rapid initiation of the entire process, including fixation, incubation, and imaging, significantly shortening the response time for emergency diagnoses and saving valuable time for clinical emergency assessment. This greatly improves the clinical emergency adaptability of single-sample indirect immunofluorescence detection methods. Furthermore, dynamic resource scheduling accommodates both sequential sample testing, ensuring the orderliness of routine testing processes, and prioritizes processing mechanisms, achieving efficient resource allocation and utilization and preventing idle equipment or resource waste. Simultaneously, this design can be fully integrated into automated fluorescence detection and analysis systems, seamlessly connecting with automated liquid handling, optical imaging, and AI image analysis modules without additional manual intervention in the scheduling process. This ensures seamless integration from sample entry to report generation, further enhancing efficient and independent sample processing characteristics. It improves the overall operational efficiency of the testing system while ensuring the stability and continuity of the testing process, providing flexible and efficient testing solutions for laboratories of different sizes.

[0059] Below are examples of practical applications. In some embodiments, the single-sample indirect immunofluorescence detection method is based on the single-container integration principle, utilizing a flat-bottomed container as a fixation and detection platform, and automating liquid handling and optical imaging to achieve an independent sample flow. AI algorithms process image features, quantify signals, and classify patterns to ensure accurate output. The working process is briefly described as follows: sample enters the container for fixation; automated antibody incubation and washing; transfer, detection, and imaging; AI analysis to generate a report. On the production line, the entire process can be completed in 45 minutes, and depending on the incubation status, it can be completed within 40 minutes, with no batch waiting, truly achieving on-demand testing and urgent priority.

[0060] In some embodiments, a single-use reaction container is used as the fixation and detection platform for indirect immunofluorescence detection, and an independent sample detection process is achieved by employing automated liquid handling and optical imaging, including sample fixation, reaction processing and optical detection, thus realizing workflow integration.

[0061] As an example, sample fixation involves introducing a biological sample, such as a cell suspension or tissue section, into a single-use reaction vessel and fixing the biological sample, i.e., the specimen, in the flat-bottomed detection area of ​​the single-use reaction vessel. In some embodiments, a fixative, such as methanol or paraformaldehyde, is used to uniformly coat the specimen onto the bottom wall of the single-use reaction vessel. For example, the bottom wall thickness is 0.1 mm to 0.5 mm, the transmittance is ≥95%, the surface roughness Ra is ≤50 nm, and the flatness deviation A is ≤137 nm. The fixative is dispensed using an automated pipetting mechanism and incubated at a controlled temperature to ensure uniform specimen adhesion.

[0062] As an example, the reaction process involves antibody incubation and washing within the same container, with all operations automated. In some embodiments, an automated pipette dispenses a first antibody for binding to the sample antigen into a single-use reaction container, incubates for 10 to 15 minutes, and then dispenses a fluorescently labeled second antibody, incubating for the same time. Unbound antibodies are automatically washed away by aspirating and dispensing buffer, leaving the sample on the bottom wall. Each step is timed independently to avoid batch variations.

[0063] As an example, optical detection involves transferring the container to the detection module after the immune reaction and using an inverted microscope to collect fluorescence signals from the bottom wall. In some embodiments, the microscope lens is positioned below the single-dose reaction container towards the bottom wall, and the focus is adjusted to capture a high-resolution image. Integrated filters and a high-sensitivity camera capture the signal. The image is transmitted to a control computer, where a large artificial intelligence model automatically calculates the fluorescence intensity or titer and identifies the karyotype, such as homogeneous or speckled.

[0064] The described single-sample indirect immunofluorescence detection method is highly integrated, fully automated from loading to result output. The single-sample reaction container is transferred between functional modules via a robotic arm. As an example, the single-sample reaction container is labeled with a barcode for tracking. The control unit dynamically schedules the process to ensure continuous processing. The workflow includes: sample loading, fixation, reaction, cleaning, incubation, detection, AI analysis, and report generation, and supports priority access for urgent samples.

[0065] Compared with the traditional indirect immunofluorescence method, this embodiment has the following significant advantages.

[0066] Shorten testing time: Individual testing is timed separately for each person, allowing for immediate testing upon arrival. The incubation time is 10 to 15 minutes, and the entire process can be completed in 45 minutes, overcoming batch delays and improving emergency diagnostic response.

[0067] Eliminate human error: Fully automated integrated container processing and AI interpretation reduce contamination, loss, and subjective differences, ensuring consistent and reliable results.

[0068] Enhanced flexibility and throughput: Supports continuous random sampling and on-demand processing of individual samples, improving laboratory adaptability and throughput. The current production line can process 50 to 100 samples per hour or even more.

[0069] Enhanced scalability: Compatible with existing systems, can be cascaded with other analyzers, simplifies infrastructure, and is suitable for laboratories of all sizes.

[0070] Improved accuracy: AI-powered automatic titer and karyotype identification handle complex samples, ensuring high accuracy and repeatability, with an accuracy rate exceeding 95%.

[0071] As an example, a convenient, fast, and cost-effective single-sample indirect immunofluorescence detection device, referred to as the detection device, is presented below. This device can be used to detect autoantibodies in autoimmune diseases. Compared to traditional indirect immunofluorescence detection techniques using glass slides as the matrix carrier, this embodiment can be manufactured into single-sample test reagents or kits, avoiding waste at both the production and client ends, reducing costs, and improving utilization efficiency. With appropriate automated instruments, samples can be tested immediately upon arrival, and even a single sample can be tested, offering the advantages of convenience and speed.

[0072] The carrier is a single-use reaction container, including but not limited to enzyme-labeled plate reaction tubes, chemiluminescent reaction tubes, other commercially available single or multiple reaction tubes, and custom-made single reaction tubes. The carrier mainly provides solid-phase support for the matrix and provides a container for liquid reactions.

[0073] The matrix used in the testing is generally cells or tissue. Cells cultured in vitro are grown on treated carriers or slides for a certain period of time, and then fixed and dried to make a cell matrix; frozen animal tissues are cut by freezing, and the tissue slices are pasted onto treated carriers or slides, and then fixed and dried to make a tissue matrix.

[0074] Cell matrix and / or tissue matrix, after being processed by instruments or manually, are made into matrix slices of millimeters in size. A certain amount of UV-curing adhesive is pre-dropped onto the bottom of the reaction tube. Then, one or more matrix slices are placed at the bottom of the reaction tube according to a certain arrangement rule. After being irradiated with ultraviolet light for a certain period of time, the adhesive is cured, thus fixing the matrix to the bottom of the reaction tube. Generally, the prepared matrix reaction tubes, together with the desiccant, are placed in an aluminum foil bag, heat-sealed, and stored for later use.

[0075] Other reagents required for the test mainly include sample diluent, phosphate buffer or powder, Tween 20, fluorescein isothiocyanate (FITC), labeled goat anti-human IgG, and mounting medium.

[0076] As an example, the detection procedure is described below. The sample is diluted and mixed according to a specific dilution ratio. Then, a certain amount of the diluted sample and control are added to each carrier, such as a reaction tube. After reacting at room temperature for 30 minutes, each reaction tube is washed and soaked with a certain amount of phosphate-Tween 20 buffer for a certain period of time. Next, a certain amount of fluorescein isothiocyanate and labeled goat anti-human IgG solution are added to each reaction tube. After reacting at room temperature for 30 minutes, each reaction tube is washed and soaked with a certain amount of phosphate-Tween 20 buffer for a certain period of time. Finally, a certain amount of sealing agent is added to each tube, thus completing the detection reaction. The reaction results are then observed using a fluorescence microscope.

[0077] Taking the detection of antinuclear antibodies using HEp-2 cell matrix as an example. HEp-2 cells cultured in vitro are seeded at a specific density onto treated glass slides and grown for approximately 24 hours. After cell fixation and drying, HEp-2 cell matrix is ​​prepared. The HEp-2 cell matrix is ​​then cut into 2mm × 2mm slices using appropriate instruments or manually. These slices are then adhered to the bottom of a carrier, such as an ELISA plate reaction tube, using UV-curable adhesive. One slice is placed in each reaction tube, and the adhesive is cured by UV irradiation for 1 minute, thus completing the matrix reaction tube. This matrix reaction tube can be used to dilute and test human serum samples. Specific fluorescence patterns observed under a fluorescence microscope can indicate possible antinuclear antibody types and related autoimmune diseases.

[0078] Taking the detection of antinuclear antibodies using HEp-2 and monkey liver matrix as an example. Frozen monkey liver tissue was cut into thin slices using a cryostat, then mounted onto a prepared glass slide, and subsequently fixed and dried to prepare the monkey liver tissue matrix. The HEp-2 cell matrix was prepared in the same manner. The HEp-2 cell matrix and monkey liver tissue matrix were cut into 1.5mm × 1.5mm slices using appropriate instruments or manually. These slices were then glued to the bottom of a carrier, such as an ELISA plate reaction tube, using UV-cured adhesive. Each reaction tube contained one HEp-2 cell matrix slice and one monkey liver tissue matrix slice. After irradiation with UV light for 1 minute to solidify the adhesive, the matrix reaction tube was complete. This matrix reaction tube can be used to dilute and test human serum samples. Specific fluorescence patterns observed under a fluorescence microscope can indicate the possible type of antinuclear antibody and related autoimmune diseases.

[0079] Taking the detection of anti-double-stranded DNA antibodies using *Clerodendrum brevis* matrix as an example, externally cultured *Clerodendrum brevis* are inoculated onto treated glass slides at a specific inoculation density. After drying, fixation, and further drying, a *Clerodendrum brevis* matrix is ​​prepared. The matrix is ​​then cut into 2mm × 2mm slices using appropriate instruments or manually. These slices are then adhered to the bottom of a carrier, such as a chemiluminescence reaction tube, using UV-curing adhesive. One slice is placed in each reaction tube, and the adhesive is cured by UV irradiation for 1 minute, thus completing the matrix reaction tube. This matrix reaction tube can be used to dilute and test human serum samples. The presence or absence of specific fluorescence under a fluorescence microscope indicates whether the anti-double-stranded DNA antibody is positive, which can be used as an auxiliary diagnostic tool for systemic lupus erythematosus.

[0080] Taking the detection of anti-keratin antibodies in rat esophageal matrix as an example, frozen rat esophageal tissue is cut into thin slices using a cryostat and then mounted onto a prepared glass slide. After fixation and drying, the rat esophageal matrix is ​​prepared. The rat esophageal matrix is ​​then cut into 2mm × 2mm slices using appropriate instruments or manually. These slices are then adhered to the bottom of a carrier, such as a chemiluminescence reaction tube, using UV-curable adhesive. One slice is placed in each reaction tube, and the adhesive is cured by UV irradiation for 1 minute, thus completing the matrix reaction tube. This matrix reaction tube can be used to detect diluted human serum samples. The presence or absence of specific fluorescence under a fluorescence microscope indicates whether anti-keratin antibodies are positive, which can be used for the early diagnosis of rheumatoid arthritis.

[0081] Taking the detection of anti-neutrophil cytoplasmic antibodies (ACNA) in human neutrophil matrix as an example, neutrophils isolated from human blood are seeded onto treated glass slides at a specific seeding density. After culture for an appropriate time, they are fixed and dried using different fixatives to prepare ethanol-fixed and methanol-fixed neutrophil matrixes, respectively. The ethanol-fixed and methanol-fixed neutrophil matrixes are then cut into 1.5mm × 1.5mm slices using appropriate instruments or manually. These slices are then glued to the bottom of a carrier, such as a custom-made single-response tube, using UV-curable adhesive. Each reaction tube contains one ethanol-fixed and one methanol-fixed neutrophil matrix slice. After irradiation with UV light for 1 minute to solidify the adhesive, the matrix reaction tube is complete. This matrix reaction tube can be used to detect diluted human serum samples. The presence and specific fluorescence pattern observed under a fluorescence microscope determine whether the anti-neutrophil cytoplasmic antibody is positive, which can be used for the diagnosis of ANCA-associated vasculitis.

[0082] Taking the detection of anti-mitochondrial antibodies, anti-smooth muscle antibodies, and anti-parietal cell antibodies in rat kidney and gastric matrix as an example, frozen rat kidney and gastric tissues are cut into thin slices using a cryostat and then mounted onto prepared glass slides. After fixation and drying, rat kidney and gastric matrix are prepared. The rat kidney and gastric matrix are then cut into 1.5mm × 1.5mm slices using appropriate instruments or manually. These slices are then glued to the bottom of a carrier, such as a custom-made single-response tube, using UV-curable adhesive. Each reaction tube contains one rat kidney matrix slice and one rat gastric matrix slice. After irradiation with UV light for 1 minute to solidify the adhesive, the matrix reaction tube is complete. This matrix reaction tube can be used to dilute human serum samples for testing. The presence and specific fluorescence patterns observed under a fluorescence microscope determine whether anti-mitochondrial antibodies, anti-smooth muscle antibodies, and anti-parietal cell antibodies are positive, which can be used to diagnose diseases such as autoimmune liver disease and autoimmune gastritis.

[0083] As can be seen from the above examples, the single-sample indirect immunofluorescence detection method and its related single-sample indirect immunofluorescence detection device can be applied to various corresponding indirect immunofluorescence detections.

[0084] In some embodiments, a single-sample indirect immunofluorescence detection system includes a single-sample reaction container, an indirect immunofluorescence processing unit, and an analysis unit. The single-sample reaction container serves as the fixation and detection platform for indirect immunofluorescence detection. The indirect immunofluorescence processing unit employs automated liquid handling and optical imaging to achieve an independent sample detection process. The analysis unit is used for automated processing of image features, signal quantification, and pattern classification output. This single-sample indirect immunofluorescence detection system can serve as a convenient, fast, and cost-effective single-sample detection device for detecting autoantibodies in autoimmune diseases. By introducing the single-sample detection concept and utilizing a dedicated single-sample reaction container and automated process, sample fixation, reaction processing, and optical detection are integrated within a single single-sample reaction container. It supports continuous random sampling, independent timing, and dynamic scheduling, achieving full automation from sample input to result output, thus improving efficiency and accuracy. In some embodiments, the single-sample indirect immunofluorescence detection system is implemented based on the single-sample indirect immunofluorescence detection method described in any embodiment; that is, the single-sample indirect immunofluorescence detection system adopts the single-sample indirect immunofluorescence detection method described in any embodiment. Since the single-sample indirect immunofluorescence detection method described in any embodiment is used, the single-sample indirect immunofluorescence detection system also has the beneficial technical effects of the single-sample indirect immunofluorescence detection method, which will not be elaborated here. As an example, the single-sample indirect immunofluorescence detection system has functional modules corresponding to each step of the single-sample indirect immunofluorescence detection method. Each functional module can be set separately or integrated. The embodiments of this application do not impose additional limitations on this.

[0085] It should be noted that other embodiments of this application also include a single-person indirect immunofluorescence detection method and system formed by combining the technical features of the above embodiments.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the patent protection scope of this application should be determined by the appended claims.

Claims

1. A single-sample indirect immunofluorescence detection method, characterized in that, Including the following steps: Single-use reaction containers were used as a fixation and detection platform for indirect immunofluorescence assays. An automated liquid handling and optical imaging process is used to achieve independent sample detection. It automatically processes image features, quantizes signals, and classifies patterns for output.

2. The single-sample indirect immunofluorescence detection method according to claim 1, characterized in that, Using a single-use reaction container as a fixation and detection platform for indirect immunofluorescence detection includes: using a single-use reaction container to contain a sample, and using the single-use reaction container containing the sample as a fixation and detection platform for indirect immunofluorescence detection, for adding reagents and carrying out the reaction.

3. The single-sample indirect immunofluorescence detection method according to claim 1, characterized in that, An automated liquid handling and optical imaging process enables independent sample detection, including: The single-use reaction vessel is transported to a fixed position, and a fixation reagent is added to the single-use reaction vessel containing the sample to achieve sample fixation. The single-use reaction container is delivered to the reaction location, and reaction reagents are added to the single-use reaction container containing the sample to achieve antibody incubation and washing. A single-use reaction vessel is transported to the detection location, and a fluorescence image is acquired from the bottom of the single-use reaction vessel.

4. The single-sample indirect immunofluorescence detection method according to claim 3, characterized in that, Each single-use reaction container is identified, and the single-use reaction container is transported to the fixed position, reaction position, and detection position using a continuous random sampling and sequential detection method; or, a first-in-first-out combined with priority identification method is used to transport the single-use reaction container to the fixed position, reaction position, and detection position.

5. The single-sample indirect immunofluorescence detection method according to claim 3, characterized in that, The single-use reaction vessel is a single-use flat-bottomed reaction vessel, which is used to acquire fluorescence images from the bottom of the single-use reaction vessel.

6. The single-sample indirect immunofluorescence detection method according to claim 3, characterized in that, The system employs automated liquid handling and optical imaging to achieve independent sample detection processes, including independent timing and / or measurement for each individual process.

7. The single-sample indirect immunofluorescence detection method according to any one of claims 1 to 6, characterized in that, Automated image feature processing, including the use of large artificial intelligence models to automatically identify and process image features obtained from optical imaging.

8. The single-sample indirect immunofluorescence detection method according to claim 7, characterized in that, Quantizing signals includes quantifying image features using standardized processing methods, calculating fluorescence intensity, and providing digital grading results.

9. The single-sample indirect immunofluorescence detection method according to claim 8, characterized in that, The classification mode output includes standard classification based on nuclear homogeneity, nuclear granules, nucleolarity, and cytoplasmic type, which is combined with an artificial intelligence model for auxiliary interpretation and output of a test report.

10. A single-sample indirect immunofluorescence detection system, characterized in that, include: Single-use reaction containers serve as a fixation and detection platform for indirect immunofluorescence assays; The indirect immunofluorescence processing unit employs automated liquid handling and optical imaging to achieve an independent sample detection process. as well as The analysis unit is used to automatically process image features, quantize signals, and classify pattern outputs.

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