A kind of automatic pretreatment and smear staining system for tuberculosis sputum sample

Abstract

The application provides a kind of whole automatic pretreatment and smear staining system of tuberculosis sputum specimen, and relates to the technical field of specimen processing.The whole automatic pretreatment and smear staining system of tuberculosis sputum specimen includes specimen receiving and bar code identification module, specimen quality automatic evaluation module, automatic liquefaction inactivation module, automatic centrifugal bacterium aggregation module, automatic smear module, automatic drop dyeing and staining module, automatic drying module, waste liquid and waste automatic treatment module and central control unit arranged in sequence:the specimen quality automatic evaluation module adopts image recognition technology combined with viscosity detection technology to grade evaluate the quality of sputum specimen;the automatic centrifugal bacterium aggregation module adopts gradient centrifugation technology.By realizing whole process closed automation, eliminating occupational exposure risk;Gradient centrifugation improves the detection rate;Standardized operation improves the consistency of results, automatic quality control, greatly improves the detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of specimen processing technology, specifically to a fully automated pretreatment and smear staining system for tuberculosis sputum specimens. Background Technology

[0002] Tuberculosis (TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis and remains a major global public health problem. Sputum smear acid-fast staining microscopy, with its advantages of simplicity, low cost, and high specificity, has been listed by the World Health Organization as the preferred rapid diagnostic method for TB and is a core tool for detecting patients with infectious pulmonary tuberculosis.

[0003] For a long time, the pretreatment and staining of sputum specimens for tuberculosis in clinical laboratories in my country have mainly relied on manual operation, which has many insurmountable drawbacks: the utilization rate of manual smear samples is only about 5%, and early or follow-up specimens with low bacterial counts are very easy to miss; the entire process involves multiple steps, including specimen reception, liquefaction and inactivation, centrifugation, and smearing, all of which require manual intervention, with a single specimen processing time exceeding 30 minutes and an average daily processing volume of less than 40 specimens per person, which is difficult to meet the needs of large-scale testing; operators have direct contact with infectious specimens, resulting in a very high risk of aerosol exposure; smear thickness, staining time, etc. are greatly affected by subjective factors, resulting in poor reproducibility of results; and there is no way to automatically screen unqualified specimens, with about 20% to 30% of saliva specimens being ineffectively processed, wasting resources and easily producing false negatives. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a fully automated pretreatment and smear staining system for tuberculosis sputum specimens, which solves the problems of low sample utilization, poor efficiency, high biosafety risks, poor result repeatability, and lack of quality control.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a fully automated pretreatment and smear staining system for tuberculosis sputum specimens, comprising, in sequence, a specimen receiving and barcode recognition module, an automatic specimen quality assessment module, an automatic liquefaction and inactivation module, an automatic centrifugation and bacterial aggregation module, an automatic smear module, an automatic drip staining module, an automatic drying module, an automatic waste liquid and waste treatment module, and a central control unit. The automatic specimen quality assessment module employs image recognition technology combined with viscosity detection technology to grade and assess the quality of sputum specimens. This module includes a high-definition industrial camera, a ring light source, a viscosity sensor, and an image processing unit. The high-definition industrial camera is used to acquire image information of the sputum specimens, the viscosity sensor is used to detect the viscosity value of the sputum specimens, and the image processing unit analyzes the images based on a deep learning algorithm, and, combined with the viscosity detection results, classifies the specimens into three levels: qualified specimens, specimens awaiting re-examination, and unqualified specimens. The automatic centrifugal microbial aggregation module uses gradient centrifugation technology, and the centrifugal force satisfies the formula: Where F is the centrifugal force, and the unit is g. This is the centrifugal radius, in cm. Rotational speed, in r / min; The automatic staining module employs a glass slide upright constant-temperature staining technique to achieve fully automated acid-resistant staining. The module includes a staining solution storage bottle, a precision peristaltic pump, a dropping head, a constant-temperature heating platform, and a waste liquid collection tank. The precision peristaltic pump accurately controls the amount and speed of the staining solution added. The temperature of the constant-temperature heating platform is controlled at 25–30°C, and the distance between the dropping head and the glass slide is 5–10 mm. The automatic waste liquid and waste treatment module includes a waste liquid collection bottle, an exhaust gas filtration device, and a medical waste collection box; the waste liquid collection bottle is used to collect waste liquid generated during the dyeing process, the exhaust gas filtration device uses a HEPA high-efficiency filter to filter the air in the system, and the medical waste collection box is used to collect used centrifuge tubes, coating rods, and other disposable consumables. The modules are connected in four ways: mechanical, pneumatic, hydraulic, and electrical. The mechanical connection is achieved through a sealed linear guide rail transfer channel between the workstations of each module. The entire cavity adopts a sheet metal sealing structure, and rubber sealing strips are installed at the joints of each module. The pneumatic connection is achieved by a HEPA high-efficiency filter unit located at the exhaust end of the top of the cavity, which is connected to the branch air channels of each workstation through the main ventilation pipeline to form a unified negative pressure exhaust system. The hydraulic connection is achieved by connecting the automatic liquid addition mechanism, precision peristaltic pump, and waste liquid collection bottle through medical-grade silicone tubing. Each staining solution storage bottle is independently connected to the drip head, and the liquid connection connector adopts a standard Luer interface. The electrical connection is achieved by the central control unit communicating with the drive motors, sensors, and solenoid valves of each module through a CAN bus. Each module has an independent control sub-board, and photoelectric position sensors are installed at the entry and exit points of each module as status handshake nodes.

[0006] Preferably, the automatic liquefaction and inactivation module includes a specimen tube clamping mechanism, an automatic liquid addition mechanism, a vortex oscillation mechanism, and a constant temperature heating mechanism; the automatic liquid addition mechanism can accurately add 2 to 4 times the volume of the specimen digestion and inactivation solution, the vortex oscillation mechanism has an oscillation frequency of 1500 to 2500 r / min and an oscillation time of 10 to 15 minutes, and the constant temperature heating mechanism has a heating temperature of 37±1℃.

[0007] Preferably, the automatic centrifugation and bacterial aggregation module includes an automatic pipetting mechanism, a centrifuge rotor, a centrifuge tube clamping mechanism, and an automatic pouring mechanism; the automatic pipetting mechanism can accurately aspirate 3-5 mL of liquefied sample into centrifuge tubes, the centrifuge rotor can simultaneously hold 12-24 centrifuge tubes, and the automatic pouring mechanism can automatically pour out the supernatant after centrifugation, retaining the bacterial precipitate at the bottom.

[0008] Preferably, the automatic coating module includes a slide storage compartment, a slide conveying mechanism, a bacterial solution coating mechanism, and a coating thickness control mechanism; the bacterial solution coating mechanism uses a stainless steel coating rod with a coating speed of 5-10 mm / s, and the coating thickness control mechanism controls the coating thickness within the range of 10-20 μm by adjusting the gap between the coating rod and the slide.

[0009] A fully automated method for pretreatment and smear staining of tuberculous sputum specimens includes the following steps: Step S1: Specimen Receiving and Barcode Identification: Place the specimen tube containing the sputum specimen into the specimen rack of the system. The system automatically scans the barcode and records the specimen information. Step S2: Automatic Specimen Quality Assessment: The system acquires images and detects viscosity of sputum specimens, and automatically removes unqualified specimens and issues a prompt based on the assessment results; Step S3: Automatic liquefaction and inactivation: Add digestion and inactivation solution to qualified specimens, perform vortex oscillation and constant temperature heating treatment to fully liquefy the sputum and inactivate Mycobacterium tuberculosis; Step S4: Automated centrifugation and bacterial aggregation: Aspirate the liquefied sample into a centrifuge tube and follow the formula... Set the centrifugation parameters and perform gradient centrifugation to precipitate Mycobacterium tuberculosis at the bottom of the centrifuge tube; Step S5: Automatic smear preparation: Resuspend the bacterial precipitate after centrifugation, take an appropriate amount of bacterial solution and drop it onto a glass slide, and use a spreading rod to spread it evenly to form a smear with uniform thickness; Step S6: Automatic staining: Add the initial staining solution, decolorizing solution and counterstaining solution in sequence to carry out acid-resistant staining, and keep the slide at a constant temperature during the staining process; Step S7: Automatic drying: Dry the stained slides with hot air at a temperature of 60-70°C for 5-10 minutes. Step S8: Automatic treatment of waste liquid and medical waste: The system automatically collects waste liquid and medical waste generated during the treatment process and filters and disinfects the air in the system.

[0010] Preferably, the gradient centrifugation in step S4 specifically involves: first centrifuging at a speed of 1500-2000 r / min for 3-5 minutes to remove large particulate impurities, and then centrifuging at a speed of 3500-4000 r / min for 10-15 minutes to allow Mycobacterium tuberculosis to fully precipitate.

[0011] Preferably, the acid-fast staining in step S6 specifically involves: adding 1.5–2.0 mL of auramine O primary staining solution and staining for 10–15 minutes; adding 1.0–1.5 mL of hydrochloric acid-alcohol decolorizing solution and decolorizing for 30–60 seconds; adding 1.0–1.5 mL of potassium permanganate counterstaining solution and counterstaining for 1–2 minutes; rinsing thoroughly with distilled water after each staining step.

[0012] This invention provides a fully automated pretreatment and smear staining system for tuberculosis sputum specimens. It has the following beneficial effects: 1. This invention achieves fully automated closed-loop processing of tuberculosis sputum specimens from receipt to finished slide output. Operators do not need to directly contact infectious specimens. Combined with HEPA high-efficiency air filtration and automatic disinfection and collection of waste liquids and waste, it completely eliminates the risk of aerosol exposure and fundamentally solves the occupational exposure hazards in the laboratory.

[0013] 2. This invention employs gradient centrifugation aggregation technology, which achieves efficient enrichment of Mycobacterium tuberculosis by precisely controlling centrifugation force parameters, thereby improving the bacterial recovery rate. Compared with manual smears, this significantly improves the positive detection rate of early patients with low bacterial counts and treatment follow-up specimens, effectively reducing missed detections.

[0014] 3. This invention adopts standardized and automated operation throughout the entire process, accurately controlling key parameters such as smear thickness, staining solution volume, staining time and temperature, completely eliminating subjective errors caused by manual operation, improving the consistency of test results between different batches and different specimens, and meeting the requirements for standardized diagnosis and treatment of tuberculosis.

[0015] 4. This invention, by equipping an automatic specimen quality assessment module, can automatically identify and remove approximately 20% to 30% of unqualified saliva specimens, avoiding ineffective processing; the entire process is automated, shortening the processing time for a single specimen, and the daily processing capacity can reach more than 300 specimens, fully meeting the needs of large-scale screening. Attached Figure Description

[0016] Figure 1 This is a system framework diagram of the present invention; Figure 2 This is a flowchart of the automatic specimen quality assessment process of the present invention; Figure 3 This is a flowchart of the steps of the present invention; Figure 4 This is a diagram of the multi-layered biosafety protection system of the present invention; Figure 5 This is a comparison chart of the absolute values ​​of the performance indicators of the present invention; Figure 6 This is a comprehensive performance comparison chart of the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: As Figures 1-6 As shown, this embodiment of the invention provides a fully automated pretreatment and smear staining system for tuberculous sputum specimens, including a specimen receiving and barcode recognition module, an automatic specimen quality assessment module, an automatic liquefaction and inactivation module, an automatic centrifugation and bacterial aggregation module, an automatic smear module, an automatic drip staining module, an automatic drying module, an automatic waste liquid and waste treatment module, and a central control unit arranged sequentially. The automatic specimen quality assessment module uses image recognition technology combined with viscosity detection technology to grade and assess the quality of sputum specimens. The automatic specimen quality assessment module includes a high-definition industrial camera, a ring light source, a viscosity sensor, and an image processing unit. The high-definition industrial camera is used to acquire image information of sputum specimens, the viscosity sensor is used to detect the viscosity value of sputum specimens, and the image processing unit analyzes the images based on deep learning algorithms and classifies the specimens into three levels: qualified specimens, specimens awaiting re-examination, and unqualified specimens based on the viscosity detection results. The automatic centrifugal culture module uses gradient centrifugation technology, and the centrifugal force satisfies the formula: Where F is the centrifugal force, and the unit is g. This is the centrifugal radius, in cm. The speed is measured in r / min. The automatic centrifugation and bacterial aggregation module includes an automatic pipetting mechanism, a centrifuge rotor, a centrifuge tube clamping mechanism, and an automatic pouring mechanism. The automatic pipetting mechanism can accurately aspirate 3-5 mL of liquefied sample into centrifuge tubes. The centrifuge rotor can hold 12-24 centrifuge tubes simultaneously. The automatic pouring mechanism can automatically pour out the supernatant after centrifugation, retaining the bacterial precipitate at the bottom. The centrifuge rotor adopts a standard horizontal angle rotor structure. The centrifugation radius r is the distance from the central axis of the centrifuge tube to the center of rotation, ranging from 8 to 12 cm. In this embodiment, 10 cm is preferred. This radius range is suitable for the size of a 15 mL standard disposable centrifuge tube, ensuring that the liquid level in the tube remains below a safe height during centrifugation. Based on the above centrifugation radius, combined with two-stage speed settings, the first-stage centrifugation force is approximately 200-450 g, mainly settling large particulate impurities such as epithelial cells and food residues. The second-stage centrifugation force is approximately 1200-1900 g, allowing Mycobacterium tuberculosis to fully settle to the bottom of the tube and form a dense bacterial precipitate. The automatic drip staining module adopts the glass slide upright constant temperature drip staining technology to realize the fully automated operation of acid-resistant staining. The automatic drip staining module includes a staining solution storage bottle, a precision peristaltic pump, a drip head, a constant temperature heating stage, and a waste liquid collection tank. The precision peristaltic pump can accurately control the dripping amount and dripping speed of the staining solution. The temperature of the constant temperature heating stage is controlled at 25-30℃, and the distance between the drip head and the glass slide is 5-10mm. The automatic liquefaction and inactivation module includes a specimen tube clamping mechanism, an automatic liquid addition mechanism, a vortex oscillation mechanism, and a constant temperature heating mechanism. The automatic liquid addition mechanism can accurately add 2 to 4 times the volume of the specimen digestion and inactivation solution. The vortex oscillation mechanism has an oscillation frequency of 1500 to 2500 r / min and an oscillation time of 10 to 15 minutes. The constant temperature heating mechanism has a heating temperature of 37±1℃. The automatic coating module includes a slide storage bin, a slide conveying mechanism, a bacterial solution coating mechanism, and a coating thickness control mechanism. The bacterial solution coating mechanism uses a stainless steel coating rod with a coating speed of 5–10 mm / s. The coating thickness control mechanism controls the coating thickness within the range of 10–20 μm by adjusting the gap between the coating rod and the slide. The automatic waste liquid and waste treatment module includes a waste liquid collection bottle, an exhaust gas filtration device, and a medical waste collection box. The waste liquid collection bottle is used to collect waste liquid generated during the dyeing process. The exhaust gas filtration device uses a HEPA high-efficiency filter to filter the air in the system. The medical waste collection box is used to collect used centrifuge tubes, coating rods, and other disposable consumables.

[0019] Specifically: The entire system adopts a fully enclosed modular design, with a central control unit centrally coordinating the collaborative operation of each workstation. The modules form a complete closed loop through four types of connections: Mechanically, each module workstation is connected via a sealed linear guide rail transfer channel. The specimen tube clamping mechanism and slide conveying mechanism move along the guide rail between adjacent workstations. The entire cavity uses a sheet metal sealing structure, and rubber sealing strips are installed at the joints of each module to ensure complete sealing and no leakage. In terms of airflow connections, a HEPA high-efficiency filter unit is located at the exhaust end of the top of the cavity, connected to the branch airflow of each module workstation via the main ventilation duct, forming a unified negative pressure exhaust system to ensure unidirectional airflow from the clean area to the contaminated area within the cavity. For liquid connections, the automatic liquid addition mechanism, precision peristaltic pump, and waste liquid collection bottle are connected via medical-grade silicone tubing. Each staining solution storage bottle has independent tubing leading to the drip head. The waste liquid tank is drained into a unified waste liquid collection bottle. All liquid connection connectors use standard Luer interfaces. Electrically, the central control unit connects via C... The AN bus communicates with the electrical components of each module, such as drive motors, sensors, and solenoid valves. Each module has an independent control sub-board to collect status signals. Each module has photoelectric position sensors as status handshake nodes when entering and leaving the workstation. The arrival of the specimen triggers the start of the process. After completion, a signal is sent to schedule the transfer, forming a closed-loop collaborative control. The front end of the system is equipped with a specimen receiving and barcode recognition module, which can place standard sputum specimen tubes in batches. The embedded barcode scanner automatically reads the specimen information and establishes electronic files. After the specimen is transferred to the quality assessment workstation, a ring light source provides uniform shadowless illumination. A high-definition industrial camera captures a complete image of the sputum in the specimen tube. At the same time, a viscosity sensor is inserted into the specimen tube to detect the viscosity of the fluid. The image processing unit extracts features such as sputum color, texture, and foam content based on a pre-trained deep learning model. Combined with the viscosity detection value, a comprehensive score is given, and the specimen is automatically divided into three levels: qualified, awaiting re-inspection, and unqualified. Unqualified specimens are directly transferred to a dedicated recycling area and a prompt is issued to avoid invalid processing. Qualified specimens enter the liquefaction and inactivation station, where they are vertically fixed by a specimen tube clamping mechanism. An automatic liquid dispensing mechanism precisely adds the digestion and inactivation solution at a ratio of 2–4 times the specimen volume. Subsequently, a vortex oscillation mechanism oscillates at a frequency of 1500–2500 rpm for 10–15 minutes, combined with constant temperature heating at 37±1℃, to fully liquefy the sputum and complete the inactivation of Mycobacterium tuberculosis. After liquefaction, an automatic pipetting mechanism transfers 3–5 mL of the specimen into disposable centrifuge tubes. The centrifuge rotor can hold 16 centrifuge tubes simultaneously, employing a gradient centrifugation process with strictly adhered centrifugation parameters. The formula is set to first remove large particles of impurities at a low speed, and then use a high speed to allow Mycobacterium tuberculosis to fully precipitate. After centrifugation, the automatic liquid pouring mechanism accurately pours out the supernatant, leaving only about 0.5 mL of bacterial precipitate at the bottom.

[0020] The pre-trained convolutional neural network model used in the image processing unit is a lightweight classification network architecture, consisting of three convolutional layers, two max-pooling layers, one fully connected layer, and one softmax output layer. The first convolutional layer has 16 3×3 kernels, the second has 32 3×3 kernels, and the third has 64 3×3 kernels. All pooling layers use 2×2 max pooling, and the fully connected layer contains 128 neurons. The model was trained on 5000 clinical sputum specimen images, including 2000 qualified specimens, 1500 specimens awaiting re-examination, and 1500 unqualified specimens. These were labeled by three physicians with over five years of clinical laboratory experience using a three-level annotation method, with the consensus of at least two physicians serving as the gold standard. Training employed a cross-entropy loss function and the Adam optimizer to increase the classification accuracy on the test set.

[0021] The overall specimen quality score is calculated using a weighted 100-point system, with image feature score accounting for 60% and viscosity detection score accounting for 40%. The image feature score is further subdivided into four sub-indicators: sputum color (20%), viscosity and appearance (25%), foam content (15%), and impurity content (40%). Color is scored based on its matching degree with the standard sputum color card; viscosity is determined by analyzing the degree of stringiness through texture feature analysis; foam content is calculated by calculating the area of ​​the foam region through image segmentation; and impurity content is scored by identifying the number of foreign particles such as food residue and detached cells. Viscosity detection uses a contact rotating viscosity probe, with 100–500 mPa·s as the acceptable range. Full marks are awarded within the range, and points are deducted linearly according to the degree of deviation if the range is outside the range. A comprehensive score below 60 is considered an unqualified specimen, 60–80 is considered a specimen requiring re-examination, and 80 or above is considered a qualified specimen.

[0022] After resuspension, the bacterial precipitate is pipetted in appropriate amounts and added to the center of a clean glass slide. The slide storage chamber can hold 200 standard glass slides, which are sequentially conveyed to the coating station by a transport mechanism. The stainless steel coating rod moves at a uniform speed of 5–10 mm / s. The coating thickness control mechanism adjusts the gap between the coating rod and the glass slide via a precision lead screw, precisely controlling the coating thickness within the range of 10–20 μm. After coating, the glass slides are transferred to the staining station, where a constant-temperature, upright glass slide staining technique is used. The constant-temperature heating stage stabilizes the glass slide temperature at 25–30°C. A precision peristaltic pump sequentially and quantitatively delivers each staining solution, with the dropper head 5–10 mm from the glass slide surface. The staining waste solution flows directly into the collection tank below. After staining, the glass slides are dried with hot air at 60–70°C for 5–10 minutes and then automatically output to the finished product area. The system operates in a closed loop. Waste liquid is collected in a unified collection bottle for each disinfection step. Exhaust gas is filtered through a HEPA filter before being discharged. Used disposable consumables automatically fall into a medical waste collection box. All operating parameters and status are monitored and recorded in real time by the central control unit.

[0023] The central control unit uses an embedded ARM controller with a real-time operating system. The overall scheduling logic is implemented based on a finite state machine, employing a pipeline parallel processing mode. A single batch can accommodate 24 specimens for pipeline operation. The typical processing times for each module are as follows: barcode recognition approximately 5 seconds / specimen, quality assessment approximately 15 seconds / specimen, liquefaction and inactivation approximately 12-15 minutes / batch, centrifugation and bacterial aggregation approximately 15-20 minutes / batch, smear approximately 30 seconds / slide, staining approximately 12-15 minutes / slide, and drying approximately 8 minutes / batch. Waste liquid and waste treatment are performed in parallel without occupying the main thread. The central control unit dynamically schedules the flow based on the status signals of each module. When the previous workstation is completed and the next workstation is idle, the transfer is automatically triggered. The total processing cycle for a single specimen is approximately 45-55 minutes, and the daily processing capacity at full load can reach more than 300 specimens. All operating parameters, workstation status, and specimen tracking information are recorded and stored in real time, supporting full traceability and query.

[0024] Example 2: A fully automated method for pretreatment and smear staining of tuberculous sputum specimens, comprising the following steps: Step S1: Specimen Receiving and Barcode Identification: Place the specimen tube containing the sputum specimen into the specimen rack of the system. The system automatically scans the barcode and records the specimen information. Step S2: Automatic Specimen Quality Assessment: The system acquires images and detects viscosity of sputum specimens, and automatically removes unqualified specimens and issues a prompt based on the assessment results; Step S3: Automatic liquefaction and inactivation: Add digestion and inactivation solution to qualified specimens, perform vortex oscillation and constant temperature heating treatment to fully liquefy the sputum and inactivate Mycobacterium tuberculosis; Step S4: Automated centrifugation and bacterial aggregation: Aspirate the liquefied sample into a centrifuge tube and follow the formula... Set the centrifugation parameters and perform gradient centrifugation to allow Mycobacterium tuberculosis to precipitate at the bottom of the centrifuge tube. The gradient centrifugation is as follows: first centrifuge at 1500-2000 r / min for 3-5 minutes to remove large particles of impurities, and then centrifuge at 3500-4000 r / min for 10-15 minutes to allow Mycobacterium tuberculosis to precipitate fully. Step S5: Automatic smear preparation: Resuspend the bacterial precipitate after centrifugation, take an appropriate amount of bacterial solution and drop it onto a glass slide, and use a spreading rod to spread it evenly to form a smear with uniform thickness; Step S6: Automatic staining: Add the primary staining solution, destaining solution, and counterstaining solution sequentially for acid-fast staining. Maintain a constant temperature on the slide during the staining process. The specific steps for acid-fast staining are as follows: Add 1.5–2.0 mL of auramine O primary staining solution and stain for 10–15 minutes; add 1.0–1.5 mL of hydrochloric acid-alcohol destaining solution and destain for 30–60 seconds; add 1.0–1.5 mL of potassium permanganate counterstaining solution and counterstain for 1–2 minutes; rinse thoroughly with distilled water after each staining step. Step S7: Automatic drying: Dry the stained slides with hot air at a temperature of 60-70°C for 5-10 minutes. Step S8: Automatic treatment of waste liquid and medical waste: The system automatically collects waste liquid and medical waste generated during the treatment process and filters and disinfects the air in the system.

[0025] Specifically: Standard screw-cap specimen tubes containing sputum samples are sequentially placed into a dedicated specimen rack that can hold 24 specimens. After being pushed into the system's inlet, an embedded barcode scanner automatically scans the barcode of each tube, recording data such as specimen number, sending department, and patient information into the central control system and generating a unique processing number. The specimens are then transported to the quality assessment station via a sealed transport channel. A ring light source provides uniform shadowless illumination, and a high-definition industrial camera captures a complete side image of the sputum inside the specimen tube. Simultaneously, a contact viscosity sensor is inserted into the specimen tube to detect the fluid's shear viscosity. The image processing unit extracts characteristic parameters such as sputum color, viscosity, foam ratio, and impurity content based on a pre-trained convolutional neural network model. Combined with the viscosity detection value, a comprehensive score is calculated. Specimens with a score below 60 are automatically transferred to a dedicated sealed recycling box. The system simultaneously issues a non-compliance warning on the operation interface and records the reason. Specimens with scores between 60 and 80 are stored separately for re-examination, while qualified specimens with scores above 80 proceed to the next processing step.

[0026] The qualified specimen is vertically fixed by the clamping mechanism. The automatic liquid addition mechanism precisely adds N-acetylcysteine-sodium hydroxide digestion and inactivation solution at a ratio of 3 times the specimen volume. Subsequently, the vortex oscillation mechanism oscillates at a frequency of 2000 r / min for 12 minutes, combined with constant temperature heating at 37℃, to fully hydrolyze and liquefy the mucoprotein in the sputum, and simultaneously complete the inactivation treatment of Mycobacterium tuberculosis. After liquefaction, 4 mL of the specimen is transferred to a disposable sterile centrifuge tube by a precision pipette tip. The centrifuge rotor adopts a standard centrifugation radius of 10 cm. First, it is centrifuged at 1800 r / min for 4 minutes to remove large particles such as epithelial cells and food residues from the sputum. After the automatic liquid pouring mechanism precisely pours out the supernatant, it is centrifuged at 3800 r / min for 12 minutes to fully precipitate Mycobacterium tuberculosis at the bottom of the centrifuge tube. Finally, about 0.5 mL of bacterial pellet is retained. The bacterial pellet is repeatedly resuspended by pipetting 3-5 times with the pipette tip.

[0027] After resuspension, 20 μL of the bacterial suspension is dropped onto the center of a clean glass slide. A stainless steel coating rod moves laterally at a uniform speed of 8 mm / s. The thickness control mechanism adjusts the gap between the coating rod and the slide to 15 μm via a precision lead screw, forming an elliptical slide with uniform thickness. After the slide is prepared, it is transferred to the staining station. A constant temperature heating table stabilizes the slide temperature at 28°C. A precision peristaltic pump sequentially delivers the staining solution in quantitative increments. The dropper head is 7 mm from the slide surface. First, 1.8 mL of auramine O primary staining solution is added for 12 minutes. After rinsing with distilled water for 5 seconds, 1.2 mL of hydrochloric acid-alcohol destaining solution is added for 45 seconds. After rinsing again, 1.2 mL of potassium permanganate secondary staining solution is added for 1.5 minutes. The staining waste liquid from each step flows directly into the sealed collection tank below. The stained slides are dried with hot air at 65°C for 8 minutes to ensure they are completely dry and free of watermarks before being automatically output to the finished slide rack. The system operates under closed negative pressure throughout. Waste liquid generated at each stage is collected in a dedicated waste liquid bottle, disinfected with chlorine-containing disinfectant, and then discharged. Air within the system is filtered through a HEPA high-efficiency filter before being discharged. Used disposable centrifuge tubes, pipette tips, coating swabs, and other consumables automatically fall into a sealed medical waste collection box. Parameters, time, and results of all processing steps are automatically recorded and archived by the central control system, allowing for traceability and query at any time.

[0028] Example 3: Automation level of processing Completely manual intervention, no automation. Automating a single step (centrifugation or staining) The entire process from specimen reception to slide output is automated. Specimen quality control None; all non-compliant specimens were processed. None; all non-compliant specimens were processed. Dual-modal evaluation combining image recognition and viscosity detection enables automatic grading and rejection. Centrifugal microbial aggregation technology Standard centrifugation, no parameter-based precise control Standard centrifugation, with fixed parameters <![CDATA[Gradient centrifugation, the centrifugal force precisely satisfies F = 1.118×10 -5 ×r×n 2 > Smear technology Hand-coated, uneven thickness Mechanical coating, without precise thickness control Precision gap control ensures the coating thickness remains stable at 10-20 μm. Staining techniques Hand-dyeing / immersion dyeing, parameters are arbitrary Immersion method, reagents shared Upright glass slides are stained at a constant temperature; individual glass slides are stained independently. Biosafety protection Open-air operation poses an extremely high risk of aerosol exposure. Semi-closed operation still poses an exposure risk Fully enclosed negative pressure operation, HEPA high-efficiency filtration, no human contact. Processing time per specimen 30-40 minutes About 20 minutes 8 minutes Maximum daily processing volume ≤40 servings / person ≤100 servings / person ≥300 servings / person Sample utilization rate Approximately 5% Approximately 20% ≥90% Consistency of results Approximately 60% Approximately 80% ≥98% Reagent consumption Approximately 25 mL / specimen Approximately 15 mL / specimen Approximately 5 mL / sample The fully automated, closed-loop process achieves complete isolation between operators and infectious specimens, reaching BSL-3 biosafety standards. Gradient centrifugation and bacterial aggregation technology increases sample utilization to over 90%, significantly improving the positive detection rate of low-bacterial-volume specimens. Standardized automated operations ensure 98% result consistency while reducing reagent consumption by 80% and increasing daily processing capacity to more than seven times that of manual operations, fully meeting the needs of clinical laboratories and large-scale tuberculosis screening.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fully automated pretreatment and smear staining system for tuberculous sputum specimens, comprising, in sequence, a specimen receiving and barcode identification module, an automatic specimen quality assessment module, an automatic liquefaction and inactivation module, an automatic centrifugation and bacterial aggregation module, an automatic smear module, an automatic drip staining module, an automatic drying module, an automatic waste liquid and waste treatment module, and a central control unit, characterized in that: The automatic specimen quality assessment module uses image recognition technology combined with viscosity detection technology to grade and assess the quality of sputum specimens. The automatic specimen quality assessment module includes a high-definition industrial camera, a ring light source, a viscosity sensor, and an image processing unit. The high-definition industrial camera is used to acquire image information of sputum specimens, the viscosity sensor is used to detect the viscosity value of sputum specimens, and the image processing unit analyzes the images based on deep learning algorithms and, combined with the viscosity detection results, classifies the specimens into three levels: qualified specimens, specimens awaiting re-examination, and unqualified specimens. The automatic centrifugal microbial aggregation module uses gradient centrifugation technology, and the centrifugal force satisfies the formula: Where F is the centrifugal force, and the unit is g. This is the centrifugal radius, in cm. Rotational speed, in r / min; The automatic staining module employs a glass slide upright constant-temperature staining technique to achieve fully automated acid-resistant staining. The module includes a staining solution storage bottle, a precision peristaltic pump, a dropping head, a constant-temperature heating platform, and a waste liquid collection tank. The precision peristaltic pump accurately controls the amount and speed of the staining solution added. The temperature of the constant-temperature heating platform is controlled at 25–30°C, and the distance between the dropping head and the glass slide is 5–10 mm. The automatic waste liquid and waste treatment module includes a waste liquid collection bottle, an exhaust gas filtration device, and a medical waste collection box; the waste liquid collection bottle is used to collect waste liquid generated during the dyeing process, the exhaust gas filtration device uses a HEPA high-efficiency filter to filter the air in the system, and the medical waste collection box is used to collect used centrifuge tubes, coating rods, and other disposable consumables. The modules are connected in four ways: mechanical, pneumatic, hydraulic, and electrical. The mechanical connection is achieved through a sealed linear guide rail transfer channel between the workstations of each module. The entire cavity adopts a sheet metal sealing structure, and rubber sealing strips are installed at the joints of each module. The pneumatic connection is achieved by a HEPA high-efficiency filter unit located at the exhaust end of the top of the cavity, which is connected to the branch air channels of each workstation through the main ventilation pipeline to form a unified negative pressure exhaust system. The hydraulic connection is achieved by connecting the automatic liquid addition mechanism, precision peristaltic pump, and waste liquid collection bottle through medical-grade silicone tubing. Each staining solution storage bottle is independently connected to the drip head, and the liquid connection connector adopts a standard Luer interface. The electrical connection is achieved by the central control unit communicating with the drive motors, sensors, and solenoid valves of each module through a CAN bus. Each module has an independent control sub-board, and photoelectric position sensors are installed at the entry and exit points of each module as status handshake nodes.

2. The fully automated pretreatment and smear staining system for tuberculous sputum specimens according to claim 1, characterized in that: The automatic liquefaction and inactivation module includes a specimen tube clamping mechanism, an automatic liquid addition mechanism, a vortex oscillation mechanism, and a constant temperature heating mechanism. The automatic liquid addition mechanism can accurately add 2 to 4 times the volume of the specimen digestion and inactivation solution. The vortex oscillation mechanism has an oscillation frequency of 1500 to 2500 r / min and an oscillation time of 10 to 15 minutes. The constant temperature heating mechanism has a heating temperature of 37±1℃.

3. The fully automated pretreatment and smear staining system for tuberculous sputum specimens according to claim 1, characterized in that: The automatic centrifugation and bacterial aggregation module includes an automatic pipetting mechanism, a centrifuge rotor, a centrifuge tube clamping mechanism, and an automatic pouring mechanism. The automatic pipetting mechanism can accurately aspirate 3-5 mL of liquefied sample into centrifuge tubes. The centrifuge rotor can simultaneously hold 12-24 centrifuge tubes. The automatic pouring mechanism can automatically pour out the supernatant after centrifugation, retaining the bacterial precipitate at the bottom.

4. The fully automated pretreatment and smear staining system for tuberculous sputum specimens according to claim 1, characterized in that: The automatic coating module includes a slide storage compartment, a slide conveying mechanism, a bacterial solution coating mechanism, and a coating thickness control mechanism. The bacterial solution coating mechanism uses a stainless steel coating rod with a coating speed of 5-10 mm / s. The coating thickness control mechanism controls the coating thickness within the range of 10-20 μm by adjusting the gap between the coating rod and the slide.

5. A fully automated method for pretreatment and smear staining of tuberculous sputum specimens, comprising the fully automated pretreatment and smear staining system for tuberculous sputum specimens according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step S1: Specimen Receiving and Barcode Identification: Place the specimen tube containing the sputum specimen into the specimen rack of the system. The system automatically scans the barcode and records the specimen information. Step S2: Automatic Specimen Quality Assessment: The system acquires images and detects viscosity of sputum specimens, and automatically removes unqualified specimens and issues a prompt based on the assessment results; Step S3: Automatic liquefaction and inactivation: Add digestion and inactivation solution to qualified specimens, perform vortex oscillation and constant temperature heating treatment to fully liquefy the sputum and inactivate Mycobacterium tuberculosis; Step S4: Automated Centrifugation and Culturing: Transfer the liquefied sample to a centrifuge tube and follow the formula... Set the centrifugation parameters and perform gradient centrifugation to precipitate Mycobacterium tuberculosis at the bottom of the centrifuge tube; Step S5: Automatic smear preparation: Resuspend the bacterial precipitate after centrifugation, take an appropriate amount of bacterial solution and drop it onto a glass slide, and use a spreading rod to spread it evenly to form a smear with uniform thickness; Step S6: Automatic staining: Add the initial staining solution, decolorizing solution and counterstaining solution in sequence to carry out acid-resistant staining, and keep the slide at a constant temperature during the staining process; Step S7: Automatic drying: Dry the stained slides with hot air at a temperature of 60-70°C for 5-10 minutes. Step S8: Automatic treatment of waste liquid and medical waste: The system automatically collects waste liquid and medical waste generated during the treatment process and filters and disinfects the air in the system.

6. The fully automated pretreatment and smear staining method for tuberculous sputum specimens according to claim 5, characterized in that: The gradient centrifugation in step S4 specifically involves: first centrifuging at 1500–2000 r / min for 3–5 minutes to remove large particulate impurities, and then centrifuging at 3500–4000 r / min for 10–15 minutes to allow Mycobacterium tuberculosis to precipitate fully.

7. The fully automated pretreatment and smear staining method for tuberculous sputum specimens according to claim 5, characterized in that: The acid-fast staining in step S6 specifically involves: adding 1.5–2.0 mL of auramine O primary staining solution and staining for 10–15 minutes; adding 1.0–1.5 mL of hydrochloric acid-alcohol decolorizing solution and decolorizing for 30–60 seconds; adding 1.0–1.5 mL of potassium permanganate counterstaining solution and counterstaining for 1–2 minutes; rinsing thoroughly with distilled water after each staining step.

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