Fully automated immunofluorescence staining slide system and method

Abstract

This invention discloses a fully automated immunofluorescence staining system and method, relating to the field of staining machine technology. The system includes an operating table equipped with a central control system and, through signal interaction with the central control system, a sample processing module, a reagent loading module, a cleaning needle module, a mounting module, a fluorescence imaging module, and an antigen loading module. The sample processing module is configured with several independent sample needle modules, each equipped with an independent Z-axis drive mechanism and an independent Y-axis drive mechanism. The central control system coordinates the operation of all modules, forming a fully automated closed loop from sample scanning, dilution, incubation, cleaning, secondary antibody loading to mounting, fluorescence imaging, and preliminary interpretation. This completely eliminates the limitations of traditional manual operation and the fragmented functionality of existing equipment, eliminating the need for manual coordination between experimental steps, significantly reducing reliance on manual labor, and effectively improving the detection efficiency of large batches of samples.

Description

Technical Field

[0001] This application relates to the field of staining machine technology, and more specifically, to a fully automated immunofluorescence staining system and method. Background Technology

[0002] In the fields of biotechnology and clinical testing, indirect immunofluorescence is a commonly used immunoassay technique, widely applied to the detection of biomarkers such as autoantibodies, providing crucial evidence for the diagnosis and monitoring of autoimmune diseases. This experiment involves multiple consecutive steps, including sample dilution, loading, incubation, washing, reagent loading, mounting, and result interpretation. Traditional methods rely on manual, step-by-step completion, resulting in cumbersome procedures, long processing times, poor reproducibility of results, high risk of cross-contamination, and subjective result interpretation.

[0003] Existing fully automated staining equipment has significant shortcomings: incomplete functional coverage, with most only automating certain experimental steps and failing to form a complete closed-loop process, still requiring manual intervention; inadequate anti-contamination design, with cleaning processes often employing single-needle washing or immersion cleaning, failing to remove residual liquids in real time; insufficient positioning accuracy, with sample rack positioning relying on conventional encoders and lacking error compensation mechanisms; and fixed cleaning paths, unable to be precisely optimized for stain distribution. These issues make existing equipment insufficient to meet the demands of rapid and accurate detection of large batches of samples in clinical settings, necessitating a technological solution. Summary of the Invention

[0004] The main purpose of this application is to provide a fully automated immunofluorescence staining system and method to solve the problems of existing equipment lacking a closed-loop process, insufficient positioning accuracy, and poor cleaning effect.

[0005] To achieve the above objectives, this application provides the following technical solution:

[0006] A fully automated immunofluorescence staining system includes: an operating table, wherein the operating table is equipped with a central control system and a sample processing module, a reagent loading module, a washing needle module, a mounting module, a fluorescence imaging module, and an antigen loading module that are signal-interacted with the central control system;

[0007] The sample processing module includes a sample placement area, a dilution well array, and several sets of independent sample needle modules. Each set of independent sample needle modules is equipped with an independent Z-axis drive mechanism and an independent Y-axis drive mechanism. The independent sample needle module is a sample needle assembly.

[0008] The sample rack in the sample placement area is equipped with an encoder, which is linked with the dynamic error compensation algorithm of the central control system to make the positioning accuracy of the sample rack ≤ ±1μm; the sample placement area has several sample positions.

[0009] The sample needle assembly has a dual-mode liquid surface detection function of capacitive + optical, and is configured with adaptive switching logic: when the capacitive detection detects bubbles or liquid surface fluctuations ≥2mm, it automatically switches to optical detection, with a detection response time ≤10ms.

[0010] The dilution pore array has a dilution pore structure consisting of a 60° cone-shaped flow guide bottom wall and an upper hydrophilic / lower hydrophobic coating. The hydrophilic coating is made of polyethylene glycol and the hydrophobic coating is made of fluorosilane.

[0011] The sample processing module is also equipped with a diluent injection structure shared with the sample needle assembly. The diluent injection structure includes a diluent level, a peristaltic pump module, and a pump. The peristaltic pump module has a built-in flow sensor and forms a closed-loop control with the central control system. The diluent delivery error is ≤±2%, and the timing linkage error with the pump is ≤0.1s.

[0012] The cleaning needle module is a multi-position dual-needle collaborative cleaning structure with independent X / Y / Z axis control. The central control system has a built-in intelligent cleaning path planning algorithm based on stain recognition by a fluorescence imaging module. It can automatically increase the number of cleaning cycles by 1 to 2 based on the area where the stain area accounts for ≥3% in the fluorescence image. The cleaning fluid temperature is controlled at 37±1℃. The aspiration needle has a built-in optical residue detection unit, and the turbidity ≤5NTU is used as the standard for judging whether the cleaning is qualified. A cleaning steel needle position is provided next to the cleaning needle module for storing and positioning the cleaning needle.

[0013] The antigen tablet loading module is equipped with an elastic adaptive clamping component with a clamping force of 0.5-2N, which can accommodate antigen tablets of three different sizes: 13×75mm, 13×100mm, and 25×75mm. The antigen tablet loading module is linked with the temperature control module of the central control system. During the incubation process, the temperature fluctuation of the antigen tablet area is ≤±0.5℃. It is also linked with the barcode scanning module integrated into the cleaning needle module through signal binding to realize automatic identification of antigen tablet information.

[0014] The system is configured with a full-duplex LIS data interaction interface, uses SSL / TLS1.3 encrypted transmission and CRC32 data verification, supports HL7v2.x / FHIR dual protocol compatibility, and has a bidirectional data interaction latency of ≤50ms;

[0015] The central control system coordinates the operation of all modules to form a fully automated closed loop from sample scanning, dilution, incubation, cleaning, secondary antibody loading to mounting, fluorescence imaging and preliminary interpretation. It achieves sequential linkage of sample positioning, injection and cleaning through intelligent scheduling algorithms.

[0016] Optionally, the reagent placement area of ​​the reagent loading module has 16 reagent positions arranged in a ring array, and each reagent position is equipped with a reagent presence and remaining quantity detection sensor at the bottom; the reagent needle and sample needle assembly of the reagent loading module share the same needle body structure, and its dispensing mode is to draw 250μl of reagent at one time and continuously add 10 times, with a maximum coverage of 50 wells in a single dispensing.

[0017] A fully automated immunofluorescence staining method, implemented based on any of the systems described above, includes the following steps:

[0018] S1: Manually load the antigen sheet into the elastic adaptive clamping assembly of the antigen sheet loading module, place the sample tube on the sample rack of the sample processing module, place the reagent bottle in the reagent position of the reagent loading module, fill the diluent into the diluent position of the diluent injection structure, and fill the cleaning solution into the corresponding container.

[0019] S2: Select or edit multiple experimental items through the central control system, set corresponding preset dilution ratios (5-200 times selectable) for different experimental items, set step-by-step dilution instructions based on near-infrared concentration detection feedback for high-concentration samples, and set step jump instructions according to the experimental sample status or preset requirements. The step jump instructions support skipping completed or unnecessary incubation and cleaning steps. The jump logic is triggered by the preset program of the central control system.

[0020] S3: After the system starts, the sample rack is located by an encoder combined with a dynamic error compensation algorithm. Based on the blockchain timestamp, a unique association match between the sample and the antigen slide information is completed. The matching response time is ≤100ms and the matching accuracy is ≥99.9%.

[0021] S4: After confirming the sample liquid level using the dual-mode liquid level detection function of the sample needle assembly, the sample is gradually diluted using the peristaltic pump module and the aspiration pump. During the dilution process, the dilution ratio is adjusted by near-infrared concentration detection feedback, and the dilution error is ≤±5%. A reciprocating motion combined with a 500rpm rotation is used for the mixing method, and the uniformity CV value is ≤3% after at least 3 blows. 25μl of diluted sample is added to the small reaction area according to the antigen sheet reaction area specifications, and 70μl of diluted sample is added to the large reaction area. The number of the independently Z / Y axis driven sample needle modules is 5 sets, the dilution time for 300 samples does not exceed 40 minutes, and the sample addition time for 300 wells after dilution does not exceed 15 minutes.

[0022] S5: After incubating the antigen tablets at room temperature for 30±2 min, the antigen tablets are cleaned using a multi-well dual-needle collaborative cleaning structure with independent X / Y / Z axis control. During the cleaning process, the cleaning path is planned based on the stain recognition results of the fluorescence imaging module. The injection of cleaning medium at 37±1℃ into the dispensing needle and the aspiration of liquid by the aspiration needle are carried out simultaneously, with a timing linkage error of ≤0.1s. The cleaning operation includes soaking in washing solution for 5 min + rinsing with purified water once. The temperature control accuracy of the washing solution is ±1℃. The cross-contamination rate of the antigen tablets after cleaning is ≤0.05%. After cleaning, the turbidity is detected by the optical residue detection unit built into the aspiration needle (≤5NTU is considered qualified), and the cleaning needle returns to the cleaning steel needle position.

[0023] S6: Add 25 μl of fluorescent secondary antibody to the small reaction area of ​​the antigen sheet according to the corresponding dose, and add 70 μl of fluorescent secondary antibody to the large reaction area. Avoid generating air bubbles during the addition process. After incubating the antigen sheet at room temperature for 30 ± 2 min, repeat the cleaning operation in step S5. The sample addition time for 300-well fluorescent secondary antibody should not exceed 15 minutes.

[0024] S7: The mounting module automatically adds mounting medium to complete the mounting process. The mounting medium is added in a mode where 200 μl of mounting medium is drawn at a time by a 5-sample needle assembly and continuously added to 20 wells. The mounting time for 300 wells is ≤10 min. The fluorescence imaging module acquires fluorescence images. The central control system outputs preliminary interpretation results of positive, negative or suspicious by quantifying fluorescence intensity and fluorescence distribution range parameters. The consistency of the interpretation is ≤3% compared with the manual standard. After the mounting is completed, the experimental data is uploaded through the full-duplex LIS data interaction interface with a transmission rate ≥10 Mbps and an abnormal retransmission rate ≤0.1%. Throughout the process, all modules work together in coordination through the intelligent scheduling algorithm of the central control system without interfering with each other. It supports parallel detection of multiple projects, and the overall detection time for 300 well samples does not exceed 2.5 hours.

[0025] The fully automated immunofluorescence staining system and method provided by this invention have the following advantages compared with the prior art:

[0026] Significantly improved positioning accuracy: By linking the encoder with the dynamic error compensation algorithm, the positioning accuracy of the sample holder reaches ≤±1μm, which solves the problem of large errors in conventional positioning methods and ensures accurate sample injection position.

[0027] The dual-mode liquid level detection function, combined with adaptive switching logic, has a detection response time of ≤10ms, effectively avoiding detection blind spots caused by bubbles and liquid level fluctuations, and preventing empty suction or excessive suction.

[0028] The special structural design of the dilution well and the precise blowing method ensure that the mixing uniformity CV value is ≤3%. Five independent sample needle modules enable rapid dilution and loading of 300 samples, greatly improving the detection throughput.

[0029] Intelligent cleaning path planning and dual-needle collaborative cleaning mode, combined with a residue detection unit, reduce the cross-contamination rate to ≤0.05% and significantly improve the cleaning qualification rate.

[0030] The flexible adaptive clamping assembly is compatible with three sizes of antigen tablets, and the temperature control linkage design ensures that the incubation temperature fluctuation is ≤±0.5℃, guaranteeing the repeatability of the test.

[0031] With dual-protocol compatibility and encrypted transmission design, the two-way data interaction latency is ≤50ms, meeting the docking needs of different hospital LIS systems and ensuring data security.

[0032] Improved efficiency through a closed-loop process: The central control system coordinates the collaborative operation of all modules, and the overall testing time for 300-well samples is ≤2.5 hours, completely eliminating reliance on manual labor and meeting the needs of high-throughput clinical testing. Attached Figure Description

[0033] The accompanying drawings, which form part of this application, are used to provide a further understanding of the application and to make other features, objects, and advantages of the application more apparent. The illustrative embodiments and descriptions of this application are used to explain the application and do not constitute an undue limitation of the application. In the drawings:

[0034] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0035] The components include: 1. Sample needle assembly; 2. Encoder; 3. Sample holder; 4. Dilution well array; 5. Antigen slide loading module; 6. Cleaning needle module; 7. Peristaltic pump fluid module; 8. Aspiration pump; 9. Dilution fluid injection structure; and 10. Cleaning steel needle position. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0037] It should be noted that the terms "first," "second," etc., used in the specification and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion.

[0038] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated device, element, or component to having a specific orientation, or to be constructed and operated in a specific orientation.

[0039] 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.

[0040] In addition, the term "multiple" should mean two or more.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0042] Example 1

[0043] like Figure 1 As shown, a fully automated immunofluorescence staining system includes an operating table. Various functional modules are integrated on the operating table in a rational layout to ensure that the modules operate collaboratively without interference, while optimizing operational convenience and space utilization. The surface of the operating table is treated with anti-corrosion coating to suit the environment of clinical laboratories.

[0044] The central control system is equipped with a touch screen and physical operation buttons. It incorporates PLC-based control logic and intelligent scheduling algorithms, and establishes signal interaction connections with the sample processing module, reagent loading module, cleaning needle module 6, mounting module, fluorescence imaging module, and antigen slide loading module 5 via RS485, Ethernet, and other communication interfaces to achieve command issuance, status feedback, and data processing. The central control system also integrates a database storage module for storing sample information, experimental parameters, test results, and other data, supporting data traceability and export.

[0045] The intelligent scheduling algorithm, based on task priority ranking, resource conflict detection, and dynamic time-series allocation logic, breaks down each module's operation into atomic tasks, prioritizing them as: sample localization / addition > cleaning / residue detection > secondary antibody addition / sealing > fluorescence imaging / data upload. A greedy algorithm detects resource conflicts and allocates non-overlapping time windows to conflicting tasks. For example, after the sample needle completes sampling, dilution and mixing are prioritized, simultaneously triggering the cleaning needle's operation, enabling parallel workflow across multiple modules. Automatic calibration is performed after every 50 localization operations to avoid error drift over long-term use, ensuring the overall processing time for 300 wells is ≤2.5 hours.

[0046] The sample processing module is located on one side of the operating table and includes a sample placement area, a dilution well array 4, and a sample needle assembly 1. The sample placement area contains a 25×10 matrix sample rack 3 with a total of 250 sample positions, accommodating standard blood collection tubes of 13×75mm and 13×100mm sizes. The sample rack 3 is equipped with an encoder 2, which acquires position data of the sample rack 3 at a frequency of 1kHz. The central control system processes the data using a dynamic error compensation algorithm to achieve a positioning accuracy of ≤±1μm.

[0047] The dynamic error compensation algorithm is implemented through error benchmark calibration, real-time error acquisition, error model fitting, closed-loop feedback adjustment, and dynamic calibration update: During system initialization, the sample holder 3 is driven to move to three calibration points: the origin, midpoint, and endpoint, and the initial error benchmark is recorded. During the movement, the instantaneous deviation is calculated in real time, and an error prediction model Δx_pred=a×t²+b×t+c is constructed using a quadratic polynomial fitting algorithm. Based on the model, compensation commands are sent to the drive motor to adjust the speed accuracy to ±1 rpm and the start-stop timing accuracy to ±0.1 ms. The error benchmark library is automatically updated after every 50 positioning cycles. The bottom of the sample holder 3 is equipped with an electric push rod drive mechanism, which can automatically push the sample holder 3 to the scanning area and the sampling area.

[0048] The dilution well array 4 is arranged adjacent to the sample holder 3 and has 300 dilution well positions. Each dilution well has a maximum capacity of 1 ml and adopts a 60° cone angle flow guide bottom wall design. The upper layer of the well wall is a polyethylene glycol hydrophilic coating and the lower layer is a fluorosilane hydrophobic coating to avoid liquid residue.

[0049] Sample needle assembly 1 is equipped with 5 independent sample needle modules, each with an independent stepper motor-driven Z-axis and Y-axis drive mechanism, achieving a drive accuracy of 0.01mm. Made of medical-grade stainless steel, sample needle assembly 1 features capacitive and optical dual-mode liquid level detection. When capacitive detection detects bubbles or liquid level fluctuations ≥2mm, it automatically switches to optical detection, with a detection response time ≤10ms. Sample needle assembly 1 has a sampling volume range of 10-100μl, with sampling accuracy controlled within ±5%.

[0050] The sample processing module also includes a diluent injection structure 9 shared with the sample needle assembly 1, comprising a diluent level, a peristaltic pump module 7, and a suction pump 8. The diluent level stores the diluent. The peristaltic pump module 7 has a built-in flow sensor, forming a closed-loop control with the central control system. The diluent delivery range is 500-1000 μl, with a delivery error ≤ ±2%, and the timing error between the pump and the suction pump 8 is ≤ 0.1 s, achieving precise liquid transfer. The sample needle assembly 1 mixes the sample and diluent using a reciprocating motion combined with a 500 rpm rotation, achieving a mixing uniformity CV value ≤ 3% after at least three cycles.

[0051] The reagent loading module is located in the central area of ​​the operating table. Its reagent placement area has 16 reagent positions arranged in a circular array, accommodating standard reagent bottles with a base diameter of 18mm, a height of 38mm, and an inner diameter of 8mm, for loading fluorescent secondary antibodies. Each reagent position is equipped with a sensor at the bottom to monitor the position and remaining reagent level of the reagent bottle in real time. When the reagent in a bottle is depleted, the central control system automatically switches the reagent needle to a backup reagent position to ensure continuous experimentation. The reagent needle of the reagent loading module shares the same needle body structure with the sample needle assembly 1 of the sample processing module. Its dispensing mode is to draw 250μl of reagent at a time and dispense it 10 times consecutively, with a maximum coverage of 50 wells per dispensing.

[0052] The cleaning needle module 6 is located on the other side of the operating table, corresponding to the antigen sheet loading module 5. It is a multi-position dual-needle collaborative cleaning structure with independent X / Y / Z axis control. The X, Y, and Z axes are all driven by linear guides and stepper motors, ensuring high positioning accuracy and precise coverage of the entire cleaning area of ​​the antigen sheet. The central control system incorporates an intelligent cleaning path planning algorithm based on stain recognition from the fluorescence imaging module. It can automatically increase the number of cleaning cycles by 1 to 2 based on areas where the stain area accounts for ≥3% in the fluorescence image.

[0053] The intelligent cleaning path planning algorithm first segments the antigen sheet image using Gaussian filtering and the Otsu method, extracting the area, mean grayscale value, and center point coordinates of the stained region. Regions are then classified as high-priority areas based on "area ≥ 0.1 mm² and mean grayscale value ≥ 128". The A* algorithm is used to plan the path with a node spacing of 0.5 mm, ensuring full coverage of high-priority areas and key point coverage of low-priority areas. The cleaning fluid injection pressure is increased by 20% and the suction negative pressure by 15% for high-priority areas. If the optical residue detection unit detects turbidity > 5 NTU, local secondary cleaning is triggered. After each sheet is cleaned, the path planning efficiency is optimized using a gradient descent algorithm.

[0054] The cleaning needle module 6 includes a dispensing needle and a suction needle that work together. The dispensing needle is connected to the cleaning solution storage container via a pipeline, and the suction needle is connected to the waste liquid collection container via a pipeline. The cleaning solution temperature is controlled at 37±1℃ with a control accuracy of ±1℃. During cleaning, the dispensing needle injects washing solution or purified water onto the surface of the antigen sheet, while the suction needle simultaneously and in real time removes residual washing solution and reaction waste liquid, with a timing error ≤0.1s. The suction needle has a built-in optical residue detection unit, using a turbidity ≤5NTU as the standard for judging whether the cleaning is qualified. A cleaning needle slot 10 is provided next to the cleaning needle module 6 for storing and positioning the cleaning needles.

[0055] The sealing module and reagent loading module are located adjacent to each other. The sealing tablet dispensing structure is shared with sample needle assembly 1. The sealing tablet is stored in a dedicated container, and the dispensing volume can be set to 10 μl or 20 μl via the central control system. The sealing tablet dispensing uses a mode where 200 μl of sealing tablet is drawn at a time from 5 sample needle assemblies 1 and continuously dispensed into 20 wells. The sealing time for 300 wells is ≤10 min. Precise control of the moving speed and dispensing flow rate is maintained during dispensing to avoid air bubbles.

[0056] The fluorescence imaging module is integrated into the upper part of the operating panel. It uses a high-resolution CCD camera as the imaging component and is equipped with a fluorescence filter group to adapt to the imaging needs of secondary antibodies at different wavelengths. The camera's shooting angle and focal length can be adjusted via an electric adjustment mechanism to ensure clear acquisition of fluorescence images of each reaction area of ​​the antigen slide. The fluorescence imaging module is connected to the central control system, and the acquired fluorescence images are transmitted to the central control system in real time for processing by the built-in fluorescence image analysis algorithm.

[0057] The fluorescence image analysis algorithm first performs dark field correction, gamma correction, and morphological opening on the image. Then, it segments the fluorescent region using an adaptive thresholding method and extracts three core parameters: mean fluorescence intensity, uniformity of fluorescence distribution, and peak fluorescence intensity. Based on a threshold library trained on 1000 clinical samples, the following criteria are used: mean fluorescence intensity < 80 and fluorescent region area < 0.5 mm² is considered negative; mean fluorescence intensity ≥ 120 and peak fluorescence intensity ≥ 200 is considered positive; values ​​in between are considered suspicious. The consistency of interpretation with human standards has an error of ≤ 3%.

[0058] The antigen tablet loading module 5 is located in the upper-middle area of ​​the operating table. Its support structure consists of multiple independent trays, each equipped with an elastic adaptive clamping component with a clamping force of 0.5-2N, accommodating antigen tablets of three different sizes: 13×75mm, 13×100mm, and 25×75mm. Positioning protrusions are provided in the tray placement slots to ensure precise placement of the antigen tablets. The antigen tablet loading module 5 is linked to the temperature control module of the central control system, ensuring that the temperature fluctuation in the antigen tablet area during incubation is ≤±0.5℃. Simultaneously, the antigen tablet loading module 5 is linked to the barcode scanning module integrated into the cleaning needle module 6 via signal binding. The barcode scanning module is an optical barcode scanner located on the side of the cleaning needle module 6. When the tray moves the antigen tablet to the scanning area, the scanner automatically scans the barcode or QR code on the surface of the antigen tablet, identifies the antigen tablet information, and transmits it to the central control system.

[0059] The system is equipped with a full-duplex LIS data interaction interface, establishing bidirectional data interaction with the hospital's LIS system via Ethernet. It employs SSL / TLS 1.3 encrypted transmission and CRC32 data verification, supports HL7v2.x / FHIR dual-protocol compatibility, has a bidirectional data interaction latency of ≤50ms, a transmission rate of ≥10Mbps, and an abnormal retransmission rate of ≤0.1%. It can automatically obtain basic sample information and testing requirements from the LIS system. After the experiment, it automatically uploads sample information, experimental parameters, and test results to the LIS system, enabling real-time interaction and traceability of experimental data.

[0060] The central control system supports editing multiple experimental items, including AKA, APF, ANCA, dsDNA, ANA, ALD, NMDAR, AECA, etc., and can meet the simultaneous detection of at least 6 experimental items. Different experimental items can independently set corresponding parameters such as dilution ratio, sample volume, and incubation time to meet the clinical needs of parallel detection of multiple items.

[0061] Example 2

[0062] The fully automated immunofluorescence staining method of this embodiment is based on the above-mentioned fully automated immunofluorescence staining system and specifically includes the following steps:

[0063] S1: Experimental Preparation Stage. The operator first removes the antigen strips from storage and equilibrates them to room temperature (20-25°C). Then, the operator manually places each antigen strip into the elastic adaptive clamping assembly of the antigen strip loading module 5, ensuring precise contact and positioning. Ordinary blood collection tubes containing the serum or plasma samples to be tested are placed on the sample rack 3 of the sample processing module, arranged in the matrix order of rack 3. Reagent vials containing fluorescent secondary antibodies are placed in the reagent positions of the reagent loading module, ensuring proper placement and accurate sensor detection. Diluent is added to the diluent filling position of the diluent filling structure 9, purified water is added to the washing solution storage container, and special sealing tablets are added to the sealing tablet container, completing the replenishment of each liquid.

[0064] S2: Parameter Setting Stage. Operators select or edit experimental items through the touch screen of the central control system, choosing at least 6 items to be detected simultaneously from multiple preset items, and independently setting the corresponding dilution ratio for each item (selectable from 5-200 times). For samples with known high concentrations, the system sets stepwise dilution instructions based on near-infrared concentration detection feedback, such as first diluting at a 1 / 10 ratio, then performing a second dilution at a 1 / 20 ratio, until the appropriate detection concentration is reached. If there are samples that have been diluted in advance or special experimental needs that do not require a certain incubation step, the system can set step jump instructions, preset the steps to be skipped, and the jump logic is triggered by the preset program of the central control system.

[0065] The stepwise dilution adjustment algorithm for near-infrared concentration detection feedback uses 940nm near-infrared light as the detection source. During system initialization, a calibration curve C=k×A+b is established using three standard samples of 10ng / mL, 100ng / mL, and 1000ng / mL. The concentration is calculated by detecting the absorbance of the sample, and the amount of diluent is deduced in reverse from the target concentration. A maximum of 3 stepwise dilutions are performed to ensure that the dilution error is ≤±5%.

[0066] S3: Information Identification and Association Stage. After system startup, the system uses encoder 2 combined with a dynamic error compensation algorithm to accurately position sample rack 3. The central control system controls the electric push rod at the bottom of sample rack 3 to move sample rack 3 to the scanning area. The barcode scanner in the scanning area automatically scans the barcode on the sample tube to obtain sample information such as sample ID and patient information. At the same time, the tray of antigen strip loading module 5 moves the antigen strip to the scanning area. The barcode scanner bound to cleaning needle module 6 automatically scans the barcode or QR code on the surface of the antigen strip to obtain the type, model and specification information of the antigen strip. The central control system associates and matches sample information and antigen strip information based on blockchain timestamps. The matching response time is ≤100ms and the matching accuracy is ≥99.9%. The information is stored in the system database to establish a traceability relationship.

[0067] The blockchain timestamp association matching algorithm assigns a unique blockchain ID to each sample-antigen slide group. The sample ID, antigen slide ID, and associated timestamp are packaged into transaction data, and a data digest is generated using the SHA-256 hash algorithm and uploaded to the consortium blockchain node. Each time a module switches, the ID matching relationship is automatically verified. If a mismatch occurs, an alarm is triggered and the process is paused. After the experiment is completed, the associated data is bound to the experimental parameters and stored for a storage period of ≥10 years. The entire process can be traced using any ID.

[0068] S4: Sample dilution and loading stage. The central control system controls the five independent sample needle modules of the sample processing module to operate synchronously according to the preset experimental parameters. Each sample needle module moves to the corresponding sample position via Z-axis and Y-axis drive mechanisms. After confirming the sample liquid level through the dual-mode liquid level detection function, the sample needle assembly 1 accurately aspirates the sample at a preset sampling volume of 10-100μl, and then moves to the corresponding dilution well position of the dilution well array 4 to inject the sample into the dilution well. Next, the sample needle assembly 1 switches to the dilution solution injection mode, and in conjunction with the peristaltic pump liquid module 7, automatically aspirates 500-1000μl of dilution solution according to the preset dilution ratio and injects it into the same dilution well. The sample and dilution solution are uniformly mixed by reciprocating up and down motion combined with a 500rpm rotation (at least 3 times), and the uniformity of mixing (CV value) is ≤3%. After mixing, the sample needle assembly 1 moves to the corresponding antigen sheet reaction area of ​​the antigen sheet loading module 5, adds 25μl of diluted sample to the small reaction area, and adds 70μl of diluted sample to the large reaction area. During the addition process, the needle tip of the sample needle assembly 1 is 1-2mm away from the surface of the reaction area, and the addition is slow to avoid the generation of air bubbles. In this step, the dilution time for 300 samples should not exceed 40 minutes, and the loading time for the diluted samples from 300 wells should not exceed 15 minutes.

[0069] S5: First Incubation and Cleaning Stage. After sample addition, the central control system controls the antigen slide loading module 5 to keep the antigen slide in a horizontal position and incubates it at room temperature for 30±2 minutes. During incubation, the system monitors the ambient temperature in real time to ensure temperature stability. After incubation, the central control system controls the cleaning needle module 6 to start, which moves the dispensing needle and aspiration needle above the antigen slide through an independent X / Y / Z axis drive mechanism. The cleaning path is planned based on the stain recognition results of the fluorescence imaging module. The dispensing needle first injects washing solution into the surface of the antigen slide, so that the antigen slide is completely immersed in the washing solution and left to stand for 5 minutes. Then, the dispensing needle switches to injecting purified water at 37±1℃, while the aspiration needle simultaneously removes the residual washing solution and purified water, completing one purified water rinse. The timing error of the dispensing and aspiration actions is ≤0.1s. After cleaning, the turbidity is detected by the optical residue detection unit built into the aspiration needle. ≤5NTU is considered qualified, and the cleaning needle returns to the cleaning needle position 10. The cross-contamination rate of the antigen slide after cleaning is ≤0.05%.

[0070] S6: Fluorescent secondary antibody loading, second incubation, and cleaning stage. After cleaning, the central control system switches sample needle assembly 1 to reagent dispensing mode, moves it to the corresponding reagent position on the reagent loading module, detects the level of the fluorescent secondary antibody using the dual-mode liquid level detection function, and draws the fluorescent secondary antibody according to the preset amount. Then, it moves the needle above the antigen slide reaction area, adding 25 μl of fluorescent secondary antibody to the small reaction area and 70 μl to the large reaction area. During the addition process, the needle tip is moved slowly to avoid air bubbles. After the secondary antibody loading is completed, the operator wipes away excess liquid around and on the reverse side of the antigen slide with paper. The central control system controls the antigen slide to continue incubating at room temperature for 30 ± 2 min. After the second incubation, the central control system controls cleaning needle module 6 to repeat the cleaning operation of step S5. In this step, the loading time for the 300-well fluorescent secondary antibody should not exceed 15 minutes.

[0071] S7: Mounting, Imaging, and Result Output Stage. After the second cleaning, the operator wipes off excess liquid from the surface of the antigen slide with paper. The central control system activates the mounting module, and sample needle assembly 1 switches to the mounting medium dispensing mode. The operator draws up the pre-set 10μl or 20μl dispensing volume of the mounting medium and moves it above the reaction area of ​​the antigen slide, precisely dispensing the mounting medium into the reaction area. No additional coverslip is needed to complete the mounting. The mounting time for 300 wells is ≤10min. After mounting, the central control system moves the fluorescence imaging module above the antigen slide, adjusts the camera's shooting angle and focus, and acquires fluorescence images of each reaction area. The acquired fluorescence images are transmitted to the central control system in real time. The central control system processes the fluorescence images using a built-in fluorescence image analysis algorithm and outputs preliminary interpretation results: positive, negative, or suspicious. Simultaneously, the system automatically uploads sample information, experimental parameters, fluorescence image data, and preliminary interpretation results to the hospital's LIS system via a full-duplex LIS data interaction interface, enabling real-time interaction and traceability of experimental data. Throughout the entire experimental process, each module operates collaboratively through the intelligent scheduling algorithm of the central control system without interfering with each other, and the overall detection time for 300 well samples does not exceed 2.5 hours.

[0072] By combining the above-mentioned hardware structure and method steps, this invention realizes the full automation of the indirect immunofluorescence assay from sample processing to result interpretation, effectively solving many defects of traditional manual operation and existing equipment, significantly improving experimental quality and detection throughput, and meeting the actual application needs of clinical laboratories.

[0073] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A fully automated immunofluorescence staining system, characterized in that, The system includes an operating table, which is equipped with a central control system and a sample processing module, a reagent loading module, a cleaning needle module (6), a sealing module, a fluorescence imaging module, and an antigen loading module (5) that are connected to the central control system via signal interaction. The sample processing module includes a sample placement area, which is equipped with a sample rack (3). The sample placement area is equipped with an encoder (2). The encoder (2) is linked with the dynamic error compensation algorithm of the central control system to make the positioning accuracy of the sample rack ≤ ±1μm. The sample processing module also includes a dilution hole array (4), a sample needle assembly (1), and a diluent injection structure (9). The diluent injection structure (9) includes a dilution level, a peristaltic pump module (7), and a pump (8). The sample needle assembly (1) has capacitive and optical dual-mode liquid level detection function. The dilution holes of the dilution hole array (4) are 60° cone-shaped flow guide bottom walls. The upper layer of the hole wall is a polyethylene glycol hydrophilic coating, and the lower layer is a fluorosilane hydrophobic coating. The cleaning needle module (6) includes a liquid dispensing needle and a liquid suction needle that cooperate with each other. The liquid dispensing needle is connected to the cleaning fluid storage container through a pipeline, and the liquid suction needle is connected to the waste liquid collection container through a pipeline. The central control system has a built-in intelligent cleaning path planning algorithm based on stain recognition by the fluorescence imaging module. The intelligent cleaning path planning algorithm extracts the stain area, gray mean, and center point coordinates by segmenting the antigen sheet image through Gaussian filtering and Otsu's method. The intelligent cleaning path planning algorithm divides the area ≥0.1mm² and gray mean ≥128 into high priority areas, and uses the A* algorithm to plan the path. The high priority areas are fully covered, and the low priority areas are covered by key points. The cleaning fluid spray pressure is increased by 20% and the liquid suction negative pressure is increased by 15% for the high priority areas. The cleaning fluid temperature is controlled at 37±1℃. The liquid suction needle has a built-in optical residue detection unit. The optical residue detection unit detects turbidity >5NTU and triggers local secondary cleaning. The antigen loading module (5) is equipped with an elastic adaptive clamping component, and the system is configured with a duplex LIS data interaction interface; The central control system coordinates the operation of all modules to form a fully automated closed loop from sample scanning, dilution, incubation, cleaning, secondary antibody loading to mounting, fluorescence imaging and preliminary interpretation.

2. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The reagent loading module has 16 reagent positions arranged in a ring array in the reagent placement area. Each reagent position is equipped with a reagent presence and balance detection sensor at the bottom. The reagent dispensing mode of the reagent loading module is to aspirate 250 μl of reagent at a time and add it 10 times continuously, with a maximum coverage of 50 wells in a single dispensing.

3. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The dual-mode liquid level detection function of the sample needle assembly (1) is configured with adaptive switching logic: when the capacitive detection detects bubbles or liquid level fluctuations ≥2mm, it automatically switches to optical detection with a detection response time ≤10ms.

4. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The sample needle assembly (1) mixes the sample and diluent by a reciprocating up-and-down motion combined with a 500 rpm rotation, and the uniformity CV value after at least 3 blows is ≤3%.

5. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The elastic adaptive clamping component of the antigen loading module (5) has a clamping force of 0.5-2N, which can be adapted to three different specifications of antigen tablets: 13×75mm, 13×100mm, and 25×75mm. The antigen loading module (5) is linked with the temperature control module of the central control system, and the temperature fluctuation of the antigen tablet area during the incubation process is ≤±0.5℃.

6. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The full-duplex LIS data interaction interface uses SSL and TLS1.3 protocols for encrypted transmission and CRC32 data verification. It supports HL7v2.x and FHIR protocols, and the bidirectional data interaction latency is ≤50ms.

7. The fully automated immunofluorescence staining system according to claim 1, characterized in that, The timing error of the liquid dispensing needle and the liquid aspiration needle of the cleaning needle module (6) is ≤0.1s; the intelligent cleaning path planning algorithm can automatically increase the number of cleaning times by 1 to 2 based on the area where the stain area accounts for ≥3% in the fluorescence image.

8. A fully automated immunofluorescence staining method, characterized in that, The system implementation based on any one of claims 1-7 includes the following steps: The system is started, and the sample rack is located by the encoder (2) and dynamic error compensation algorithm. The unique association and matching of sample and antigen sheet information is achieved by the blockchain timestamp. After the sample needle component (1) is detected by the dual-mode liquid level, it works with the peristaltic pump liquid module (7) and the suction pump (8) to complete the stepwise dilution of the sample. The dilution ratio is adjusted by near-infrared concentration detection, and the dilution error is ≤±5%. After mixing, the corresponding volume of sample is injected into the different reaction zones of the antigen sheet. After the sample is added, the central control system controls the antigen sheet loading module (5) to keep the antigen sheet in a horizontal state and incubates it at room temperature for 30±2 minutes. During the incubation, the system monitors the ambient temperature in real time. After the incubation, the central control system controls the cleaning needle module (6) to start, which moves the dispensing needle and the suction needle to the top of the antigen sheet. The cleaning path is planned based on the stain recognition result of the fluorescence imaging module. The cleaning operation includes soaking in the washing solution for 5 minutes and rinsing with purified water once. The temperature of the washing solution is maintained at 37±1℃ and the precision is controlled. The temperature is ±1℃; after cleaning, the central control system controls the sample needle assembly (1) to switch to the reagent dispensing mode, moves to the corresponding reagent position of the reagent loading module, detects the liquid level of the fluorescent secondary antibody through the dual-mode liquid level detection function, and then draws the fluorescent secondary antibody according to the preset amount. Then it moves to the top of the antigen sheet reaction area, adds 25μl of fluorescent secondary antibody to the small reaction area and 70μl of fluorescent secondary antibody to the large reaction area. After the secondary antibody is added, wipe off the excess liquid around and on the back of the antigen sheet. The central control system controls the antigen sheet to continue to incubate at room temperature for 30±2min. After the second incubation, the central control system controls the cleaning needle module (6) to repeat the cleaning operation. After the second cleaning, wipe the excess liquid on the surface of the antigen sheet with paper. The central control system controls the sealing module to start, and the sample needle assembly (1) switches to the sealing tablet dispensing mode. Draws the sealing tablet according to the preset dispensing amount, and then moves to the top of the antigen sheet reaction area to accurately drop the sealing tablet into the reaction area.

9. The fully automated immunofluorescence staining method according to claim 8, characterized in that, The sealing tablet was added using a 5-sample needle assembly (1) to aspirate 200 μl of sealing tablet at a time and continuously add it to 20 wells. The sealing time for 300 wells was ≤10 min. Experimental data were uploaded through a duplex LIS data interaction interface with a transmission rate ≥10 Mbps and an abnormal retransmission rate ≤0.1%.

Images