Cell counting device and method based on microfluidic technology

Abstract

The invention relates to a microfluidic technology, and particularly provides a microfluidic technology-based cell counting device and method.The cell counting device comprises a microfluidic disc, and a sample adding groove is formed in the disc; the micro-fluidic disc is also internally provided with a separation medium storage tank and a separation medium storage tank, wherein the density of the separation medium is 1.09 g / ml-1. 1g / ml; the separating tank is arranged on the outer sides of the separating medium storage tank and the sample adding tank and is respectively communicated with the sample adding tank and the separating medium storage tank; the first tiling chamber is arranged on the outer side of the separation tank and is communicated with the upper part of the separation tank, and the second tiling chamber is arranged on the outer side of the separation tank and is communicated with the lower part of the separation tank. The method has the advantages of no damage, high precision and the like.

Description

Technical Field

[0001] This invention relates to microfluidics, and more particularly to cell counting devices and methods based on microfluidics. Background Technology

[0002] A blood cell analyzer, also known as a blood cell counter, is a device specifically designed for the automated counting and classification of blood cells. Blood cell counters have a wide range of applications, covering multiple fields such as clinical diagnosis, disease monitoring, treatment monitoring, and research laboratories.

[0003] The complexity of traditional hematology analyzers also limits their application. They typically require complex sample processing and procedures, such as sample preparation, reagent addition, and instrument calibration, which not only increases the difficulty of operation but also prolongs the diagnostic time. In scenarios such as emergency care, clinical clinics, and telemedicine points, there is a need for rapid and accurate blood cell analysis results; however, the complex operating procedures of traditional hematology analyzers often fail to meet this requirement.

[0004] The emergence of miniaturized fluidized bed hematology analyzers offers a solution to the problems of traditional hematology analyzers, such as their large size and high cost. By employing a miniaturized fluidized bed system and achieving instrument compactness and portability, they enable rapid and accurate blood cell analysis in smaller medical settings such as clinics and mobile medical vehicles, thereby improving the accessibility and efficiency of medical services. However, despite significant progress in reducing instrument size and adapting to smaller medical settings, miniaturized fluidized bed hematology analyzers still face some inherent problems. First, the cumbersome operation of miniaturized fluidized bed hematology analyzers is a major issue. Although these analyzers have simplified the operation process compared to traditional hematology analyzers, they still require a series of complex steps such as sample processing, reagent addition, and instrument calibration. These operations can be confusing for untrained medical personnel, reducing the practicality and operability of the equipment. Second, miniaturized fluidized bed hematology analyzers face difficulties in maintenance and high maintenance costs. Due to their miniaturized fluidized bed system, any malfunction can only be handled by specialized technicians, which not only increases maintenance costs but may also lead to prolonged downtime, affecting the continuity and stability of diagnostic services. Furthermore, miniaturized liquid-based hematology analyzers suffer from reagent waste. Failure to fully utilize liquid reagents such as cleaning and diluents within their expiration dates leads to waste and loss, increasing diagnostic costs and operational burdens. Therefore, while miniaturized liquid-based hematology analyzers have made some progress in addressing the bulkiness of traditional analyzers, they still face inherent problems related to liquid circuits, such as inconvenience in use, maintenance difficulties, and reagent waste, making them less than ideal for point-of-care testing (POCT) hematology analyzers. To address these challenges, further technological research and development and product innovation are needed to find simpler, more efficient, and stable hematology analysis solutions to meet the healthcare industry's urgent need for rapid and accurate diagnosis.

[0005] Current methods for detecting blood cells suffer from problems such as cumbersome procedures, reliance on manual processing and inaccuracies. These issues limit the efficiency and reliability of blood cell counting. Summary of the Invention

[0006] To address the shortcomings of the existing technical solutions, the present invention provides a cell counting device based on microfluidic technology.

[0007] The objective of this invention is achieved through the following technical solution: A cell counting device based on microfluidic technology includes a microfluidic disk with a sample loading groove disposed therein; the microfluidic disk also includes: A separation medium storage tank, wherein the density of the separation medium is between 1.09 g / ml and 1.1 g / ml; A separation tank is disposed outside the separation medium storage tank and the sample loading tank, and is connected to the sample loading tank and the separation medium storage tank respectively; A first tiling chamber and a second tiling chamber, wherein the first tiling chamber is located outside the separation groove and is connected to the upper part of the separation groove, and the second tiling chamber is located outside the separation groove and is connected to the lower part of the separation groove.

[0008] Another objective of this invention is to provide a cell counting method, which is achieved through the following technical solution: A cell counting method based on the counting device of the present invention includes the following steps: A1. The disc rotates, and the blood in the sample loading tank enters the separation tank, while the separation medium in the separation medium storage tank enters the separation tank; A2. As the disc rotates, red blood cells, white blood cells, and platelets in the blood separate, with white blood cells and platelets remaining in the upper layer of the separation medium and red blood cells remaining in the lower layer of the separation medium; A3. The disc rotates, and white blood cells and platelets enter the first tiling chamber and are tiled, while red blood cells enter the second tiling chamber and are tiled. A4. Obtain images of the first and second tiled chambers, and obtain the number of white blood cells and red blood cells after image analysis.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention can automatically separate, dilute, mix, transfer, and lay out blood cells for detection, enabling blood to meet the requirements of image-based blood cell counting and classification. Automated implementation fundamentally eliminates human interference in the detection process, allowing POCT equipment to truly achieve the goal of "sample in - result out," providing a new technical path to improve the quality of blood cell detection and increasing detection efficiency. 2. This invention achieves non-destructive separation of white blood cells, platelets, and red blood cells by constructing density difference bodies using density separation media or red blood cell sedimentation solutions. On the one hand, it can enrich and detect the small number of white blood cells in the blood, improving detection accuracy. On the other hand, it can partially transfer the large number of red blood cells in the blood, reducing their dilution ratio and reducing reagent consumption. 3. By designing a temporary storage chamber and a flattening chamber, namely through a unique siphon-capillary flattening design, white blood cells, platelets, and red blood cells can be evenly and quickly spread out. Compared with traditional hemolysis or chemical separation methods, this invention does not require the addition of hemolysin or other chemical reagents, thus avoiding morphological damage and integrity loss to sensitive cells such as leukocytes and platelets. The separation process is physically non-destructive, the cells are well-intact, and the test results are closer to the true state of blood. Compared to existing microfluidic blood cell counting devices, this invention integrates an image analysis module, which automatically classifies and counts the separated blood cells through high-precision image acquisition and analysis algorithms, improving detection sensitivity and accuracy, and supporting comprehensive analysis of blood cell morphological characteristics, thus possessing higher technological advancement and application value. Attached Figure Description

[0010] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. In the drawings: Figure 1 This is a schematic diagram of the structure of a cell counting device based on microfluidic technology according to an embodiment of the present invention; Figure 2 This is a schematic diagram of separation according to an embodiment of the present invention.

[0011] In the attached diagram, 1-separation medium storage tank, 2-sample addition tank, 3-fourth flow channel, 4-fifth flow channel, 5-separation tank, 6-tenth flow channel, 7-first flow channel, 8-first diluent storage chamber, 9-sixth flow channel, 10-first dilution chamber, 11-second diluent storage chamber, 12-seventh flow channel, 13-second dilution chamber, 14-eighth flow channel, 15-dispensing tank, 16-first quantitative chamber, 17-second quantitative chamber, 18-third quantitative chamber, 19-waste tank, 20-first temporary storage chamber, 21-first spreading chamber, 22-ninth flow channel, 23-second temporary storage chamber, 24-second spreading chamber, 25-second flow channel, 26-third flow channel. Detailed Implementation

[0012] Figures 1-2 The following description illustrates optional embodiments of the invention to teach those skilled in the art how to implement and reproduce the invention. Some conventional aspects have been simplified or omitted to explain the technical solutions of the invention. Those skilled in the art should understand that variations or substitutions derived from these embodiments will be within the scope of the invention. Those skilled in the art should understand that the following features can be combined in various ways to form multiple variations of the invention. Therefore, the invention is not limited to the following optional embodiments, but is defined only by the claims and their equivalents.

[0013] Example 1.

[0014] Figure 1A schematic diagram of the structure of a cell counting device based on microfluidic technology according to an embodiment of the present invention is provided, as follows: Figure 1 As shown, it includes a microfluidic disk 61, and the judgment 61 is equipped with: Sample loading trough 2 is used to add blood samples.

[0015] Separation medium storage tank 1, wherein the density of the separation medium is between 1.09 g / ml and 1.1 g / ml.

[0016] The separation tank 5 is located outside the separation medium storage tank 1 and the sample addition tank 2, and is connected to the sample addition tank 2 and the separation medium storage tank 1 respectively.

[0017] The first flat chamber 21 is located outside the separation groove 5 and is connected to the upper part of the separation groove 5. The second flat chamber 24 is located outside the separation groove 5 and is connected to the lower part of the separation groove 5.

[0018] To achieve better separation, the separation tank 5 is further connected to the sample loading tank 2 via the fifth flow channel 4 and to the separation medium storage tank 1 via the fourth flow channel 3. When the disc 61 rotates, the time when the sample in the sample loading tank 2 enters the separation tank 5 is later than the time when the separation medium enters.

[0019] To achieve automated quantification and dilution, the microfluidic disk 61 is further provided with: The quantitative unit includes a dispensing tank 15, a waste liquid tank 19, and a quantitative chamber 16-18 that are connected. The quantitative unit is disposed between the separation tank 5 and the first flat chamber 21. The inlet of the dispensing tank 15 is connected to the upper part of the separation tank 5, and the quantitative chamber is connected to the first flat chamber 21.

[0020] The first dilution unit includes a first diluent storage chamber 8 and a first dilution chamber 10. The first dilution chamber 10 is located inside the metering unit, with its inlet connected to the upper part of the separation tank 5 and its outlet connected to the distribution tank 15.

[0021] The second dilution unit includes a second diluent storage chamber 11 and a second dilution chamber 13. The second dilution chamber 13 is disposed between the separation tank 5 and the second spreading chamber 24. The inlet is connected to the lower part of the separation tank 5 through the first flow channel 7, and the outlet is connected to the second spreading chamber 24.

[0022] To prevent excessive red blood cells from entering the second dilution chamber 13, the separation tank 5 is further connected to the second dilution chamber 13 through the first flow channel 7. From the separation tank 5 to the second dilution chamber 13, the rotation radius of the first flow channel 7 first decreases and then increases, and an exhaust hole is provided at the point where the rotation radius is the smallest.

[0023] To ensure uniform and rapid cell spread, the microfluidic disk 61 is further provided with: The first temporary storage chamber 20 has its inlet connected to the upper part of the separation tank 5, and its outlet connected to the first flat chamber 21 through the second flow channel 25; the rotation radius of the second flow channel 25 decreases from the first temporary storage chamber 20 to the first flat chamber 21.

[0024] The second temporary storage chamber 23 has its inlet connected to the lower part of the separation tank 5, and its outlet connected to the second tiling chamber 24 via the third flow channel 26; the rotation radius of the third flow channel 26 decreases from the second temporary storage chamber 23 to the second tiling chamber 24.

[0025] When the disk rotates, the liquid in the first and second temporary storage chambers is siphoned.

[0026] To further improve the separation effect, the separation medium is mixed with red blood cell sedimentation fluid.

[0027] The cell counting method based on the counting device of this embodiment includes the following methods: A1. The disc 61 rotates, and the blood in the sample loading tank 2 enters the separation tank 5, and the separation medium in the separation medium storage tank 1 enters the separation tank 5.

[0028] A2. As the disc 61 rotates, red blood cells, white blood cells, and platelets in the blood separate. White blood cells and platelets remain in the upper layer of the separation medium, while red blood cells remain in the lower layer. Figure 2 As shown.

[0029] A3. The disc 61 rotates, and white blood cells and platelets enter the first flattening chamber 21 and are flattened, while red blood cells enter the second flattening chamber 24 and are flattened.

[0030] A4. Obtain images of the first tiled chamber 21 and the second tiled chamber 24, and obtain the number of white blood cells and red blood cells after image analysis.

[0031] In order to achieve uniform and rapid spreading of cells, in step A3, leukocytes and platelets enter the first temporary storage chamber 20 and are siphoned, and enter the first spreading chamber 21 through the second flow channel 25, and spread by capillary action; from the first temporary storage chamber 20 to the first spreading chamber 21, the rotation radius of the second flow channel 25 becomes smaller.

[0032] Red blood cells enter the second temporary storage chamber 23 and are siphoned, then enter the second spreading chamber 24 through the third flow channel 26 and spread out via capillary action; from the second temporary storage chamber 23 to the second spreading chamber 24, the radius of rotation of the third flow channel 26 decreases.

[0033] Example 2.

[0034] Application examples of the cell counting device and method based on microfluidic technology according to Embodiment 1 of the present invention.

[0035] In this application example, such as Figure 1 As shown, the main body 11 is fan-shaped.

[0036] The separation medium storage tank 1 contains the separation medium and erythrocyte sedimentation solution, and is connected to the outer separation tank 5 via a (straight) fourth flow channel 3. The sample loading tank 2 is connected to the separation tank 5 via a (curved) fifth flow channel 4. Due to the curvature of the fifth flow channel 4, the blood sample takes longer to enter the separation tank 5 compared to the separation medium when the disc 61 rotates. The separation medium is Ficoll, mixed with 6% hydroxyethyl starch.

[0037] The upper part of the separation tank 5 is connected to the first dilution chamber 10 through the tenth flow channel 6. From the separation tank 5 to the first dilution chamber 10, the radius of rotation of the tenth flow channel 6 first decreases and then increases. The first diluent storage chamber 8 is connected to the first dilution chamber 10 through the straight sixth flow channel 9.

[0038] The first dilution chamber 10 is connected to the distribution tank 15 through the eighth flow channel 14. Along the flow direction, the radius of rotation of the eighth flow channel 14 first decreases and then increases. The outer side of the distribution tank 15 has three quantitative chambers. The first quantitative chamber is connected to the first temporary storage chamber 20 through a flow channel. The second quantitative chamber 17 is used for hemoglobin detection, and the third quantitative chamber 18 is used for specific protein detection.

[0039] The first temporary storage cavity 20 is connected to the first tiling chamber 21 through the second flow channel 25. From the first temporary storage cavity 20 to the first tiling chamber 21, the rotation radius of the second flow channel 25 becomes smaller.

[0040] The lower part of the separation tank 5 is connected to the second dilution chamber 13 via the first flow channel 7. From the separation tank 5 to the second dilution chamber 13, the radius of rotation of the first flow channel 7 first decreases and then increases, and it has an exhaust hole at its minimum point. The second diluent storage chamber 11 is connected to the second dilution chamber 13 via the seventh flow channel 12. The second dilution chamber 13 is connected to the second temporary storage chamber 23 via the ninth flow channel 22. From the second dilution chamber 13 to the second temporary storage chamber 23, the radius of rotation of the ninth flow channel 22 first decreases and then increases.

[0041] The second temporary storage chamber 23 is connected to the second tiling chamber 24 through the third flow channel 26. From the second temporary storage chamber 23 to the second tiling chamber 24, the rotation radius of the third flow channel 26 becomes smaller.

[0042] The cell counting method and the working method of the counting device of this embodiment of the invention include the following steps: A1. Component assembly: The blood to be tested is injected into the sample loading tank 2, the first rotation speed is started, and the density separation medium in the storage tank 1 can quickly enter the bottom of the separation tank 5 through the fourth flow channel 3. Due to the obstruction effect in the fifth channel 4, the blood enters the separation tank 5 later than the density gradient medium in the storage tank 1.

[0043] A2. Centrifugation: After the density separation medium and blood have completely flowed into the separation tank 5, the second rotation speed is started. Red blood cells, due to their higher density, will penetrate the density gradient medium during centrifugation and enter the bottom 53 of the separation tank 5. White blood cells and platelets, due to their lower density, remain in the upper layer 51 of the density gradient medium, with the medium layer 52 in the middle. This achieves non-destructive separation of different blood cell components, such as… Figure 2 As shown.

[0044] Leukocyte and platelet transfer: After the different cells in the blood are separated, the third rotation speed is started. The mixed liquid in the upper layer of the separation tank 5, including plasma, leukocytes, platelets and a small amount of density gradient medium, is transferred to the first dilution chamber 10 through the tenth flow channel 6. Then the liquid in the first dilution storage chamber 8 is released and enters the first dilution chamber 10 through the sixth flow channel 9 to mix with the transferred liquid, so as to realize the functions of dilution, staining and other processing.

[0045] Red blood cell transfer: The fourth rotation speed is briefly started, and a small number of red blood cells in the lower layer of the separation tank 5 are transferred to the second dilution chamber 13 through the first flow channel 7. Because the first flow channel 7 has an exhaust hole design at the siphon apex, the siphon stops after centrifugation and descent, so the remaining red blood cells will not be transferred to the second dilution chamber 13.

[0046] When the fifth rotation speed is started, the red blood cell diluent in the second diluent storage chamber 11 enters the second dilution chamber 13 through the seventh flow channel 12 to achieve the dilution treatment of red blood cells.

[0047] A3. White blood cell and platelet processing: Upon starting the sixth rotation speed, the cell mixture in the first dilution chamber 10 enters the distribution tank 15 via the eighth flow channel 14, and is evenly and quantitatively distributed into the first quantitative chamber 16, the second quantitative chamber 17, and the third quantitative chamber 18. Excess liquid enters the waste liquid tank 19. The liquid in the first quantitative chamber 16 is used for white blood cell and platelet distribution detection; the liquid in the second quantitative chamber 17 is used for hemoglobin detection; and the liquid in the third quantitative chamber 18 is used for specific protein detection.

[0048] When the seventh rotation speed is started, the liquid in the first metering chamber 16 breaks through the capillary valve and enters the first temporary storage chamber 20.

[0049] When the eighth rotation speed is started, the liquid in the first temporary storage chamber 20 is siphoned, and then the cells are spread out in the first spreading chamber 21 by capillary action.

[0050] Red blood cell processing: The seventh rotation speed is started, and part of the liquid in the second dilution chamber 13 enters the second temporary storage chamber 23 through the ninth flow channel 22.

[0051] When the eighth rotation speed is started, the liquid in the second temporary storage chamber 23 is siphoned, and then the cells are spread out in the second spreading chamber 24 by capillary action.

[0052] A4. Detection: The first flat chamber 21 is photographed and scanned by the detection instrument to obtain flat images of white blood cells and platelets, and the total white blood cell count and five-part differential data, as well as platelet data, are analyzed.

[0053] The second flat chamber 24 was photographed and scanned by the detection instrument to obtain the flat image of red blood cells, and the total number and typing data of red blood cells were analyzed.

[0054] Example 3.

[0055] The application example of the cell counting device and method based on microfluidic technology according to Embodiment 1 of the present invention differs from Embodiment 2 in that: Percoll was used as the separation medium.

Claims

1. A cell counting device based on microfluidic technology, comprising a microfluidic disk, wherein a sample loading groove is disposed within the disk; characterized in that, The microfluidic disk also contains: A separation medium storage tank, wherein the density of the separation medium is between 1.09 g / ml and 1.1 g / ml; A separation tank is disposed outside the separation medium storage tank and the sample loading tank, and is connected to the sample loading tank and the separation medium storage tank respectively; A first tiling chamber and a second tiling chamber, wherein the first tiling chamber is located outside the separation groove and is connected to the upper part of the separation groove, and the second tiling chamber is located outside the separation groove and is connected to the lower part of the separation groove.

2. The cell counting device according to claim 1, characterized in that, The separation tank is connected to the sample loading tank and the separation medium storage tank through a flow channel. When the disc rotates, the time when the sample in the sample loading tank enters the separation tank is later than the time when the separation medium enters.

3. The cell counting device according to claim 1, characterized in that, The microfluidic disk also contains: A metering unit, comprising a dispensing tank, a waste liquid tank, and a metering chamber connected together, wherein the metering unit is disposed between a separation tank and a first flat-laying chamber, the inlet of the dispensing tank being connected to the upper part of the separation tank, and the metering chamber being connected to the first flat-laying chamber; The first dilution unit includes a first diluent storage chamber and a first dilution chamber. The first dilution chamber is located inside the metering unit, with its inlet connected to the upper part of the separation tank and its outlet connected to the metering unit. The second dilution unit includes a second diluent storage chamber and a second dilution chamber. The second dilution chamber is located between a separation tank and a second spreading chamber. The inlet is connected to the lower part of the separation tank, and the outlet is connected to the second spreading chamber.

4. The cell counting device according to claim 3, characterized in that, The separation tank is connected to the second dilution chamber through the first flow channel. From the separation tank to the second dilution chamber, the rotation radius of the first flow channel first decreases and then increases. An exhaust hole is set at the point where the rotation radius is the smallest.

5. The cell counting device according to claim 1, characterized in that, The metering chamber includes a first metering chamber, a second metering chamber, and a third metering chamber, with the first metering chamber connected to the first tiling chamber.

6. The cell counting device according to claim 1, characterized in that, The microfluidic disk also contains: The first temporary storage chamber has its inlet connected to the upper part of the separation tank, and its outlet connected to the first tiling chamber via a second flow channel; from the first temporary storage chamber to the first tiling chamber, the rotation radius of the second flow channel decreases. The second temporary storage chamber has its inlet connected to the lower part of the separation tank and its outlet connected to the second flat chamber via a third flow channel. From the second temporary storage chamber to the second flattening chamber, the rotation radius of the third flow channel decreases; When the disk rotates, the liquid in the first and second temporary storage chambers is siphoned.

7. The cell counting device according to claim 1, characterized in that, The separation medium contains red blood cell sedimentation fluid.

8. The cell counting device according to claim 1, characterized in that, The separation medium is either Ficoll or Percoll.

9. A cell counting method based on the counting device of claim 1, wherein the cell counting method comprises the following steps: A1. The disc rotates, and the blood in the sample loading tank enters the separation tank, while the separation medium in the separation medium storage tank enters the separation tank; A2. As the disc rotates, red blood cells, white blood cells, and platelets in the blood separate, with white blood cells and platelets remaining in the upper layer of the separation medium and red blood cells remaining in the lower layer of the separation medium; A3. The disc rotates, and white blood cells and platelets enter the first tiling chamber and are tiled, while red blood cells enter the second tiling chamber and are tiled. A4. Obtain images of the first and second tiled chambers, and obtain the number of white blood cells and red blood cells after image analysis.

10. The cell counting method according to claim 9, characterized in that, In step A3, white blood cells and platelets enter the first temporary storage chamber and are siphoned, then enter the first spreading chamber through the second flow channel and spread out via capillary action; from the first temporary storage chamber to the first spreading chamber, the radius of rotation of the second flow channel decreases. Red blood cells enter the first temporary storage chamber and are drawn in by a siphon, then enter the second spreading chamber through the third channel and spread out by capillary action. From the second temporary storage chamber to the second flattening chamber, the rotation radius of the third flow channel decreases.

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