An immunodetection combined leukocyte counting synchronous detection device and a detection method thereof

Abstract

The application discloses a synchronous detection device for immunological detection and white blood cell counting and a detection method thereof, and belongs to the technical field of analysis and detection. The device provided by the application can automatically and synchronously complete immunological index detection and white blood cell counting by only dropping a blood sample to be detected into a sample adding port of a detection card, and is simple to operate and high in detection efficiency. The device is simple in structure, convenient to carry, and can be applied to bedside detection, grass-roots diagnosis and treatment, and detection of the illness of wounded and sick people in special environments. The detection method using the device has a long time window and is not disturbed by impurities, and thus the detection accuracy is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a synchronous detection device and method for combining immune detection with white blood cell counting, which can simultaneously perform immune detection and total white blood cell counting. Background Art

[0002] C-reactive protein (CRP) exists in trace amounts in a healthy body and participates in inflammatory responses throughout the body. When acute trauma, inflammation, and infection occur, the CRP concentration in the blood will rise rapidly. It is a commonly used clinical acute phase reaction indicator and can be detected 6-12 hours after the onset of inflammation. Initially, CRP testing was mainly used for the diagnosis of active rheumatic fever. Due to the limitations of the test results and the relatively low sensitivity, it was not taken seriously in its initial application. With the development of immunology and analytical biology in recent years, more sensitive, simple and accurate detection methods have gradually emerged, and CRP has gradually become a hot topic in clinical research.

[0003] With the development of laboratory medicine technology, white blood cell counts and infection immunity index tests in routine blood tests have become necessary indicators for diagnosing the cause of fever in patients. Currently, emergency departments in hospitals conduct tests on white blood cells and infection indexes for patients with fever and colds, and determine the cause of the disease based on the comprehensive test results. Among them, the white blood cell count in routine blood tests can make a preliminary diagnosis of acute infection. If the patient's white blood cell count is elevated, it is most likely a bacterial infection. However, the white blood cell count is easily affected by physiological factors, age, emotions, and other factors. Therefore, it is necessary to combine immunological indicators, such as CRP or PCT, which will also increase significantly in the event of infection. Therefore, the combination of white blood cell count and immunological indicators can better provide doctors with a quick and reliable basis for judging the cause of infection, which has clinical significance for guiding doctors to use medications correctly.

[0004] In recent years, the incidence of adverse reactions caused by the misuse of antibiotics has increased, drawing widespread public attention. Therefore, when administering antibiotics to patients, a confirmed diagnosis of bacterial infection should be performed first. Dual testing of immune indicators and white blood cell counts can provide an initial diagnosis of bacterial infection and guide clinical medication use. For patients with elevated levels of both indicators, antibiotics can be used. For patients with both indicators within the normal range, antibiotics can be omitted or reduced in use.

[0005] Currently, immune testing and white blood cell counts are generally performed separately in clinical testing, using two different instruments. This results in low efficiency, high cost, and a large demand for blood sampling. Especially in emergency situations, improving testing efficiency and speed is crucial to reducing patient waiting time and doctor workload.

[0006] Among them, the main methods for detecting white blood cell counts include the Coulter method and flow cytometry. The principle of the Coulter method is based on the non-conductive nature of blood cells. The resistance change caused by blood cells suspended in an electrolyte solution when passing through the counting aperture is the detection principle. The resistance changes caused by white blood cells, red blood cells, and platelets are different, thereby realizing the identification and counting of white blood cells. The flow cytometry method uses laser scattering and fluorescence staining technology to detect the scattered light and fluorescence of single cells one by one in the flow path to achieve white blood cell count. The testing equipment of the above method has the characteristics of fast detection speed and high efficiency, but it is expensive, large in size, and has high maintenance costs and requirements. It is suitable for clinical testing in large hospitals and is difficult to apply to bedside testing, primary diagnosis and treatment, and treatment of the wounded and sick in special environments.

[0007] In addition, there is a new type of white blood cell counting device in the prior art - HemoCue, which uses the principle of hemolysis counting: first, the red blood cells in the blood are dissolved with a hemolytic agent, and then the white blood cells are stained with methylene blue. The stained sample is placed under transmitted light. Since the white blood cells are darker in color after being stained, they will appear as dark spots on the image under the irradiation of transmitted light. The total number of white blood cells can be calculated by counting the number of dark spots. However, the hemolysis counting method used by HemoCue has the following defects: First, this method uses the transmission absorption method, which is easily interfered by impurities; second, the detection time window is short and must be strictly controlled within 2-10 minutes. If the time is too short, the red blood cells will not be completely dissolved, and if the time is too long, some white blood cells will be dissolved. The above two points lead to low detection accuracy of the device. Summary of the Invention

[0008] In order to solve one or more problems existing in the prior art, one aspect of the present invention provides a simultaneous detection device for immune detection combined with white blood cell counting, comprising:

[0009] case;

[0010] a main control board, which is disposed in the housing;

[0011] a photoelectric detection unit, which is disposed in the housing and electrically connected to the main control board, and includes an immune detection module and a total white blood cell count detection module, each detection module including an image sensor for capturing images;

[0012] a data processing unit, integrated into the main control board, for processing and analyzing images captured by the image sensor;

[0013] a human-computer interaction unit, electrically connected to the main control board; and

[0014] The detection card can be installed in the card insertion port provided on the housing for installing the detection card, and includes an immune detection sheet and a total white blood cell count detection sheet, which are respectively adapted to the immune detection module and the total white blood cell count detection module.

[0015] The above test card is a combined immune test and white blood cell count test card, including:

[0016] A detection card bottom shell (200), a detection card upper cover (200'), and an immune detection piece (300) and a total white blood cell count detection piece (400) installed in a box body formed by the detection card upper cover and the detection card bottom shell, wherein:

[0017] The detection card upper cover (200') is provided with a first sample loading hole (216) for loading a sample on the immune detection sheet (300), a second sample loading hole (217) for loading a sample on the total white blood cell count detection sheet (400), and a first interpretation window (218) for transmitting the immune detection area of ​​the immune detection sheet (300) and a second interpretation window (219) for transmitting the white blood cell detection area of ​​the total white blood cell count detection sheet (400). The detection card bottom shell (200) is provided with a third interpretation window (223) for transmitting the white blood cell detection area of ​​the total white blood cell count detection sheet (400). The second interpretation window (219) and the third interpretation window (223) are aligned. The first sample loading hole (216) and the second sample loading hole (217) are separated from each other, or connected to form a total sample loading port (1c).

[0018] The above-mentioned total white blood cell count detection sheet (400) includes an assembly handle (21) and a test cavity (22) provided on the assembly handle. The test cavity (22) is a semi-open cavity formed by two parallel cavity side walls (23) with a certain gap, and has a test area (24), an injection port (25), and a drainage area (26) connecting the injection port (25) and the test area (24). The thickness of the test area (24) is Less than the thickness of the drainage area (26) The injection port (25) is aligned with the second sample addition hole (217), and the test area (24) is aligned with the second interpretation window (219) and the third interpretation window (223).

[0019] The test cavity (22) is provided with at least one exhaust hole (71 or 72), which is a through hole connecting the interior of the test cavity with the outside atmosphere, and penetrates one cavity side wall of the test area (24) or the drainage area (26) or symmetrically penetrates two cavity side walls of the test area (24) or the drainage area (26); the test card bottom shell (200) and / or the test card upper cover (200') are provided with at least one communication hole (201-204) penetrating the box body, and the exhaust hole is connected to the outside atmosphere through the communication holes whose number and position match the exhaust hole.

[0020] The above-mentioned total white blood cell count detection sheet (400) further includes any one of the following or superimposed technical means:

[0021] Technical means 1: The sampling port (25) is located at the upper edge opening of the two cavity side walls (23) of the test cavity (22), wherein the upper edge of one of the two cavity side walls (23) located at the sampling port (25) is provided with a sampling notch (28) so as to inject the liquid sample through the sampling notch (28);

[0022] Technical means 2: The injection port (25) is in a concave arc shape, and the angle α between the tangent of the downward arc and the horizontal reference plane of the injection port (25) is in the range of 15°-45°;

[0023] Technical means three: The inner edge of the end edge of the cavity side wall (23) is provided with a transition fillet (210), and the radius R of the transition fillet value ranges from 0.2 mm to 1.5 mm.

[0024] The above-mentioned immunoassay sheet (300) comprises: an analysis membrane, a binding pad, a sample pad, and a water-absorbing pad which are overlapped and fixedly arranged on a sticky backing, wherein a detection band and a quality control band are arranged on the analysis membrane, and the detection band and the quality control band constitute the immunoassay area of ​​the immunoassay sheet.

[0025] The above-mentioned immune detection module (104) includes:

[0026] A light source plate (7) for emitting light;

[0027] a first optical filter (6) for transmitting light of a fixed wavelength range in the light emitted by the light source;

[0028] A spectroscope (5) for transmitting light emitted by the light source of the first filter and reflecting fluorescence emitted by the immunoassay sheet;

[0029] A second filter (4) is used to transmit light of a fixed wavelength range in the fluorescence emitted by the immunodetection piece reflected by the spectroscope;

[0030] A first imaging lens (3) is used to image the fluorescence emitted by the immunodetection sheet; and

[0031] A first image sensor (2) for focusing fluorescence imaging and transmitting the image to a data processing unit;

[0032] The first filter is arranged between the spectroscope and the light source, the spectroscope is arranged between the immunodetection plate and the first filter, the imaging lens is arranged at a position where it can receive the fluorescence reflected by the spectroscope, and the imaging lens is arranged between the second filter and the first image sensor;

[0033] Preferably, the light source panel (7) is composed of a plurality of LED light sources arranged in parallel.

[0034] The total white blood cell count detection module (105) includes:

[0035] a light source (13) for emitting light;

[0036] a third optical filter (12) for transmitting light of a fixed wavelength range in the light emitted by the light source;

[0037] A reflector (8) is used to reflect the fluorescence emitted by the total white blood cell count detection sheet onto the imaging lens;

[0038] A second imaging lens (9) is used to image the fluorescence emitted by the total white blood cell count detection sheet;

[0039] A fourth filter (10) for transmitting light of a fixed wavelength range in the imaging fluorescence; and

[0040] a second image sensor (11), for focusing cell fluorescence imaging and transmitting the imaging to a data processing unit;

[0041] The third filter is arranged between the total white blood cell count detection plate and the light source, the total white blood cell count detection plate is arranged between the third filter and the reflector, the second imaging lens is arranged at a position where it can receive the fluorescence reflected by the reflector, and the fourth filter is arranged between the second image sensor and the second imaging lens.

[0042] The above device also includes a printer, which is arranged in the housing and electrically connected to the human-computer interaction unit through the main control board, and is used to print out the results of the image processing and analysis performed by the data processing unit; and / or

[0043] The human-computer interaction unit includes a display screen, function keys and a switch button; and / or

[0044] The device also includes a card inserting base plate, a silicone pad, a heat dissipation port, a heat dissipation fan, a USB interface and a power interface, wherein the card inserting base plate is used for placing the detection card.

[0045] Another aspect of the present invention provides a method for performing immunoassay combined with leukocyte counting, comprising the following steps:

[0046] 1) Sample production

[0047] A blood sample is added to the total sample loading port of the test card or to the first sample loading port and the second sample loading port, respectively, so that the blood sample flows into and fills the test cavity of the total white blood cell count detection sheet and reacts with the pre-packaged reagent in the test cavity to form a white blood cell detection surface; the blood sample is allowed to flow to the reaction area of ​​the immune detection sheet and react with the immune target in the immune reaction area to form an immune detection area;

[0048] The reagent includes a cell fluorescent dye and a surfactant, wherein the cell fluorescent dye is one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, and Cy5; and the surfactant is one or more of quaternary ammonium salt, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, and NP-40.

[0049] 2) Imaging detection

[0050] Insert the test card containing the reacted sample obtained in step 1) into the card insertion port of the device, start the detection program to perform imaging detection, and use the image sensors of the immune detection module and the leukocyte detection module to respectively capture the fluorescence images of the samples in the corresponding detection areas;

[0051] 3) Image processing

[0052] The data processing unit processes the fluorescence images collected by the image sensor separately to obtain the results of immune detection and white blood cell detection;

[0053] 4) Result output

[0054] The detection results after image processing are displayed and output in the manual interaction unit and / or printed out through a printer.

[0055] The specific steps of the imaging detection in step 2) above include:

[0056] S1: The main control board drives the light source of the total white blood cell count detection module to light up. Under the excitation of the light source, the stained white blood cells in the total white blood cell count detection area emit fluorescence. The fluorescence signal is reflected by the reflector, and is imaged by the second imaging lens and the fourth filter to the second image sensor to obtain the total white blood cell count detection image. The light source is then turned off.

[0057] S2: The main control board drives the light source board of the immunoassay module to light up. Under the excitation of the light source, the fluorescence emitted by the immunoassay area is reflected by the spectroscope, and then focused by the second filter and the first imaging lens in the first image sensor to obtain an immunoassay image. The light source is then turned off.

[0058] S1 and S2 are performed synchronously or sequentially.

[0059] The specific method of image processing in step 3) above is:

[0060] For the processing of total white blood cell count detection images: Before processing, the magnification of the imaging light path is pre-calibrated with a graticule; then, based on the image, the number of fluorescent bright spots within the sample detection range is calculated. Furthermore, the cavity area of ​​the test cavity of the total white blood cell count detection strip corresponding to the detection range is calculated based on the magnification. Furthermore, based on the cavity thickness of the test cavity of the total white blood cell count detection strip, the solution volume corresponding to the detection range is calculated, and the white blood cell concentration is obtained. The calculation formula is as follows:

[0061]

[0062] in: ILNum is the final total leukocyte concentration; N is the number of fluorescent bright spots; is the dilution ratio of the blood sample; S is the chamber area of ​​the test cavity of the total white blood cell count detection piece corresponding to the detection range, in mm 2 ; d is the chamber thickness, unit: μm;

[0063] For the processing of the immunoassay image: the data processing unit identifies and calculates the total fluorescence intensity of the fluorescent bright band in the image, and calculates the concentration of the analyte in the blood sample based on the fluorescence intensity.

[0064] The synchronous detection device for immune detection combined with white blood cell counting provided by the above technical solution can simultaneously perform immune detection and white blood cell counting, and has the following beneficial effects compared with the existing technology:

[0065] 1) The test card of the present invention integrates immune detection and total white blood cell count detection functions. During the test, the blood sample to be tested only needs to be dripped into the sample injection port of the test card. The device provided by the present invention can automatically and synchronously complete the immunological index detection and total white blood cell count, which is simple to operate and improves the detection efficiency.

[0066] 2) The device of the present invention has a simple structure and is easy to carry, and can be applied to bedside detection, primary diagnosis and treatment, and condition detection of the injured and sick in special environments.

[0067] 3) The device of the present invention utilizes the basic principles of immunochromatography technology and fluorescent staining to simultaneously achieve immune detection and total white blood cell count. It has a long detection time window and is not interfered by impurities, thereby increasing the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A schematic diagram of the system structure of the synchronous detection device for immune detection combined with leukocyte count provided by the present invention;

[0069] Figure 2 A schematic diagram of the internal structure of the synchronization detection device provided by the present invention;

[0070] Figure 3 This is a schematic diagram of the assembly structure of the card inserting base plate and the detection card;

[0071] Figure 4 Frames A and B in the middle are schematic diagrams of the external structure of the synchronization detection device provided by the present invention;

[0072] Figure 5 Schematic diagram of the upper surface structure of the detection card;

[0073] Figure 5A Schematic diagram of the three-dimensional structure of the detection card;

[0074] Figure 5B The bottom surface and perspective structure diagram of the test card;

[0075] Figure 6 Schematic diagram of the structure of the immune detection piece of the test card;

[0076] Figure 7A This is a schematic diagram of the structure of the total white blood cell count detection piece;

[0077] Figure 7B for Figure 7A Middle BB cross-section;

[0078] Figure 8 It is a structural diagram of the photoelectric detection module;

[0079] Figure 9 It is a structural diagram of the light source board of the immunoassay unit;

[0080] Figure 10 Detect image for total white blood cell count;

[0081] Figure 11 Immunodetection images. DETAILED DESCRIPTION

[0082] In view of the defects of the prior art in which immune detection and total white blood cell count are performed separately, such as low detection efficiency, high cost and large blood volume requirement, the purpose of the present invention is to provide a synchronous detection device for immune detection combined with total white blood cell count.

[0083] like Figure 1Figure 1 shows a schematic diagram of the system architecture of the simultaneous detection device for immunoassay and white blood cell count provided by the present invention. The device comprises a test card, a photoelectric detection unit (including an immunoassay module 104 and a total white blood cell count detection module 105), a data processing unit, and a human-computer interface unit. The test card integrates an immunoassay zone and a total white blood cell count detection zone, allowing for simultaneous immunoassay and total white blood cell count detection. The immunoassay module primarily comprises a light source, an imaging optical path, and an image sensor. It corresponds to the immunoassay zone on the test card and is used to detect immune response signals, with the image sensor used to capture fluorescence images from immunoassay detection. The total white blood cell count detection module primarily comprises a light source, an imaging optical path, and an image sensor. It corresponds to the total white blood cell count detection zone on the test card and is used to detect total white blood cell count, with the image sensor used to capture fluorescence images from total white blood cell count detection. The data processing unit primarily performs image processing and result analysis. For immunoassay fluorescence images, the data processing unit identifies and calculates the total fluorescence intensity of bright fluorescent bands in the image and calculates the concentration of the substance being detected based on the fluorescence. For total white blood cell count images, the data processing unit counts all bright fluorescent spots in the image to obtain the total white blood cell count. The human-computer interface unit primarily comprises a display screen and function buttons, controlling the detection process and outputting results. The photoelectric detection unit, data processing unit and human-computer interaction unit are electrically connected to each other through the main control board.

[0084] like Figure 2 The figure shows a schematic diagram of the internal structure of the synchronous detection device for immunoassay combined with white blood cell count provided by the present invention, which mainly includes: a main control board 101, a second illumination light source 102, a first illumination light source 103, a first optical detection module, a second optical detection module and a card inserting base plate 106 arranged in a housing 100. The main control board 101 controls the second illumination light source 102, the first illumination light source 103, the first optical detection module and the second optical detection module to operate. The second illumination light source 102 provides a light source for total white blood cell count detection and forms a total white blood cell count detection module together with the second optical detection module. The first illumination light source 103 provides a light source for immunoassay and forms an immunoassay module together with the first optical detection module. The card inserting base plate 106 is used to place the detection card, such as Figure 3 FIG, which shows a schematic diagram of the assembly relationship between the card inserting base plate 106 and the detection card 1 (the detailed structure will be described below), combined with Figure 5 As shown, the immune detection area 1b on the detection card 1 corresponds to the first optical detection module, and the total white blood cell count detection area 1d corresponds to the second optical detection module. Figure 4Panels A and B in the middle show schematic diagrams of the external structure of the simultaneous detection device for immunoassay combined with white blood cell count provided by the present invention, which mainly includes: a housing 100, a display screen 107, a switch button 108, a card insertion port 109, a silicone pad 110, a heat dissipation vent 111 (corresponding to a heat dissipation fan provided in the housing 100, not shown in the figure), a printer 112, a power interface 113 and a USB interface 114. The display screen 107 (which can be a touch screen, including function keys) is used to display and output test results and is electrically connected to the main control board 101; the card insertion port 109 is set on the shell 100, for placing the test card, corresponding to the card insertion base plate 106, and the test card is inserted into the defined position on the card insertion base plate 106 in the instrument through the card insertion port 109 for testing; the silicone pad 110 can play a shockproof effect on the instrument to help protect the counter; the heat dissipation port 111 is set on the shell 100, and a corresponding heat dissipation fan is set in the shell 100 to reduce the ambient temperature of the instrument when it is working; the printer 112 is set in the shell 100, electrically connected to the human-computer interaction unit through the main control board 101, and can be used to print out the test results; the USB interface 114 can be used to transmit data, and the power interface 113 is used to connect to the power supply.

[0085] like Figure 5 The figure shows a schematic diagram of the structure of the test card 1 of the simultaneous detection device for immunoassay and cell counting provided by the present invention, which includes a handheld area 1a, an immunoassay area 1b, a total sample injection port 1c, and a total white blood cell count detection area 1d. The immunoassay area 1b and the total white blood cell count detection area 1d are respectively located on both sides of the sample injection port 1c. During use, the blood sample to be tested is directly added dropwise to the total sample addition port 1c. A portion of the blood sample automatically flows into the total white blood cell count detection sheet placed in the test card 1 and enters the detection area 1d, where it reacts with the reagents pre-sealed in the test cavity of the total white blood cell count detection sheet (including cell fluorescent dyes and surfactants, wherein the cell fluorescent dyes can be one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and other fluorescent dyes. The surfactant can be one or more of quaternary ammonium salts, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, NP-40 and other surfactants); another portion of the blood sample flows to the immune detection sheet placed in the test card 1, forming a detectable signal band in the immune detection area 1b.

[0086] Figure 5A and Figure 5BThe structure of the test card 1 is shown. It is a combined test card for immunoassay and white blood cell count. The test card 1 includes a test card cover 200', a test card bottom shell 200, and an immunoassay chip 300 and a total white blood cell count test chip 400 installed in a box formed by the test card cover 200' and the test card bottom shell 200. The test card cover 200' is provided with a first sample loading hole 216 for loading the immunoassay chip 300, a second sample loading hole 217 for loading the total white blood cell count test chip 400, and an analytical membrane 311 (see FIG. 1 ) facing the immunoassay chip 300. Figure 6 , which will be described in detail later) and the test area 24 (see Figure 11 The test card base 200 includes a second reading window 219 (described in detail later), and a third reading window 223. The first reading window 218 is a window hole extending through the test card top cover 200', while the second reading window 219 and the third reading window 223 are windows extending through the test card top cover 200' and the test card base 200, respectively, and the two windows are aligned. The test card integrates the immune detection strip 300 and the total white blood cell count detection strip 400 through the box structure formed by the test card base 200 and the test card top cover 200'. The first sample loading hole 216 on the test card top cover 200' faces the sample pad 313 of the immune detection strip 300, while the second sample loading hole 217 faces the sample inlet 25 of the total white blood cell count detection strip 400.

[0087] Figure 7A and Figure 7B The structure of a total white blood cell count detection chip 400 is shown. The total white blood cell count detection chip 400 is a slit microfluidic chip structure, comprising an assembly handle 21 and a test cavity 22. The front end of the assembly handle 21 extends into the test cavity 22 with an arc-shaped edge. The test cavity 22 is a semi-open cavity formed by two parallel sidewalls 23 with a certain gap. The test cavity 22 includes a sample inlet 25, a sample inlet notch 28, a test area 24, and a drainage area 26 connecting the sample inlet 25 and the test area 24. The injection port 25 is located at the upper edge opening of the two cavity side walls 23 of the test cavity 22. The upper edge of one of the two cavity side walls 23 at the injection port 25 is provided with an injection notch 28 to facilitate positioning of the pipette for adding samples. The injection port 25 is concave arc-shaped, and the angle α between the tangent of its downward arc and the horizontal reference plane of the injection port 25 is preferably in the range of 15°-45°. This angle can determine the direction of the flow of the liquid sample to be tested into the drainage area 26, ensuring that the added liquid sample flows spontaneously into the test area 24 in a predetermined manner and fills the test area 24. The test area 24 is located in the test cavity 22, and its thickness is The thickness of the test area 24 is 60μm-120μm. The liquid sample enters the test area 24 to form a white blood cell detection area. The thicker the test area, the larger the sample carrying capacity per unit area of ​​the white blood cell detection area and the greater the depth of field, which is suitable for the overall accurate measurement of the number of cells. The thinner the thickness, the larger the spread area of ​​the liquid sample of the same volume in the white blood cell detection area, which is suitable for the precise differentiation of cell types. The drainage area 26 is located in the test cavity 22 and is connected to the sample inlet 25 and the test area 24. The thickness of the drainage area 26 is 20μm-120μm. The range is generally 120μm-500μm, the thickness of the test area 24 Less than the thickness of the drainage area 26 , which can evenly and quickly introduce the flow path formed by the drainage area 26 from the sample inlet 25 into the test area 24 and fill it, and is also conducive to the discharge of bubbles. In order to prevent the liquid sample to be tested from flowing out due to gravity when the total white blood cell count detection piece 400 is moved, a transition fillet 20 is provided on the inner edge of the end of the cavity side wall 23, and the range of the transition fillet radius R is 0.2mm-1.5mm. After the test cavity 22 is filled with the sample, the liquid sample forms a stable liquid bridge surface at the transition fillet 20 at the end of the two cavity side walls 23, which can effectively balance the gravity of the liquid and prevent it from flowing out. In order to further avoid the generation of bubbles, at least one exhaust hole is provided on the test cavity 22, Figure 7A Two exhaust holes 71 and 72 are shown. The exhaust holes are through holes that connect the interior of the test cavity 22 with the outside atmosphere. The exhaust holes can be located on one side or both sides of the test area 24 or the drainage area 26. That is, the exhaust holes penetrate the side wall of one side of the test area 24 or the drainage area 26 or symmetrically penetrate the two side walls of the test area 24 or the drainage area 26. Preferably, the exhaust holes are symmetrical inverted cone-shaped through holes, see Figure 7B As shown, the exhaust holes 71 and 72 have small tapered ends opening toward the interior of the drainage area 26 , and large tapered ends opening outward to communicate with the outside atmosphere.

[0088] Corresponding to the design of the exhaust hole in the total white blood cell count detection sheet 400, it is necessary to open a connecting hole on the detection card cover 200' and / or the detection card bottom shell 200, such as Figure 5A It is shown that four communication holes 201, 202, 203 and 204 are designed on the detection card cover 200'. The number and distribution of the communication holes are preferably matched with the exhaust holes to facilitate the escape of bubbles in the test cavity 22 through the exhaust holes and the communication holes.

[0089] like Figure 6Figure 3 shows a schematic diagram of the structure of an immunoassay strip 300 within the immune reaction zone. Immunoassay strip 300 is an immunochromatographic test strip and primarily comprises an analytical membrane (primarily made of nitrocellulose) 311, a conjugate pad (primarily made of glass fiber) 312, a sample pad (primarily made of glass fiber or absorbent paper) 313, and an absorbent pad (primarily made of absorbent paper) 314. These components are adhered and fixed to an adhesive backing 315 in a certain overlapping relationship. This overlapping relationship ensures the continuity of the liquid sample flow along the test strip. During testing, a blood sample is dripped onto the total sample loading port 1c or the first sample loading port 216. The sample flows toward the sample pad 313 and then enters the conjugate pad 312 through osmosis and siphoning. The marker-bioactive molecule conjugate (e.g., CRP monoclonal antibodies labeled with fluorescein, quantum dots, fluorescent microspheres, or fluorescent latex particles, and rabbit IgG labeled with fluorescein, quantum dots, fluorescent microspheres, or fluorescent latex particles) is redissolved and released. Under the siphoning action of the absorbent pad 314, the conjugate leaves the conjugate pad 312 and enters the analytical membrane 311, where it flows toward the absorbent pad 314. During this process, a specific immune reaction will occur between the conjugate, CRP, the detection band 316 (for example, coated with another CRP monoclonal antibody at a different epitope from the CRP monoclonal antibody labeled with fluorescein, quantum dots, fluorescent microspheres or fluorescent latex particles), and the quality control band 317 (for example, coated with anti-rabbit IgG) (the conjugate on the conjugate pad 312, the coating on the detection band 316 and the quality control band 317 can be collectively referred to as immune targets), and generate an indicative signal, wherein the detection band 316 and the quality control band 317 serve as the immune detection area 1b.

[0090] like Figure 8 FIG. 1 is a schematic diagram showing the structure of the photoelectric detection unit of the synchronous detection device for immune detection combined with leukocyte counting provided by the present invention. The photoelectric detection unit is composed of an immune detection module and a leukocyte detection module, wherein:

[0091] The immune detection module mainly includes a first image sensor 2, a first imaging lens 3, a second filter 4, a spectrometer 5, a first filter 6 and a light source board 7 (corresponding to the first illumination light source 103). Figure 9As shown, the light source board 7 is composed of 4-6 parallel LED light sources, preferably SMD packaged LED light sources. The operating principle of the immune detection module is that the emission wavelength of the LED light source matches the immunoreactive fluorescent marker, that is, the LED light source wavelength is the excitation wavelength of the fluorescent marker. The illumination light emitted by the light source board 7 first passes through the first filter 6. The transmission wavelength range of the first filter 6 is less than 40nm, and the center wavelength of the transmission spectrum is the same as the center wavelength of the LED light source. After passing through the first filter 6, the illumination light is transmitted through the beam splitter 5 and irradiated onto the immune detection area 1b of the test card 1. The beam splitter 5 is a dichroic beam splitter with a transmittance greater than 90% for the illumination light emitted by the light source plate 7 and a reflectance greater than 90% for the fluorescence emitted by the immune reaction marker. The beam splitter 5 is installed at a 45° angle, so the fluorescence emitted by the immune detection area 1b of the test card 1 is deflected 90° after being reflected by the beam splitter 5. It is focused and imaged on the first image sensor 2 (which can be an area array image sensor with a resolution greater than 1 million pixels) through the second filter 4 and the first imaging lens 3. The second filter 4 is a fluorescence filter with a transmittance greater than 90% for the fluorescence emitted by the immune reaction marker and a transmittance less than 0.01% for the illumination light emitted by the light source plate 7.

[0092] The total white blood cell count detection module primarily includes a light source 13 (corresponding to the second illumination source 102), a third optical filter 12, a reflector 8, a second imaging lens 9, a fourth filter 10, and a second image sensor 11. Light source 13 is an LED and is located directly below the total white blood cell count detection area 1d on the test card 1, at a distance of 10-30 mm. Light emitted from light source 13 first passes through third optical filter 12, whose transmission spectrum has a central wavelength that matches the central wavelength of light source 13. Third filter 12 is located between the test card 1 and light source 13. The signal light emitted by white blood cells in the blood sample, after reacting with the reagent, under the illumination of light source 13, is reflected by reflector 8 and polarized 90°. It is then focused and imaged by second imaging lens 9 and fourth filter 10 onto second image sensor 11 (which can be an area array image sensor with a resolution greater than 3 million pixels).

[0093] The data processing unit processes and analyzes images captured by the image sensor. For immunoassay fluorescence images, the data processing unit identifies and calculates the total fluorescence intensity of bright fluorescent bands within the image, and calculates the concentration of the substance being tested based on the fluorescence. For total white blood cell count images, the unit counts all fluorescent bright spots in the image, each representing a white blood cell, and calculates the total white blood cell count. This yields the immunoassay and total white blood cell count results, respectively. The main control board controls the data processing unit to display the test results on the human-computer interface unit's display screen. Users can transfer and print the test results as needed.

[0094] The following is a more detailed description of the present invention with reference to specific embodiments, and further elaboration of the present invention, but is not intended to limit the present invention.

[0095] Example 1: Simultaneous detection device for immune detection combined with white blood cell count

[0096] The synchronous detection device for immune detection combined with white blood cell count provided in this embodiment includes a housing, a main control board, a detection card, a photoelectric detection unit (including an immune detection module and a total white blood cell count detection module), a data processing unit, and a human-computer interaction unit. The main control board is disposed within the housing, and the photoelectric detection unit, the data processing unit, and the human-computer interaction unit are electrically connected to the main control board. The detection card, the data processing unit, and the human-computer interaction unit are as described above in conjunction with the accompanying drawings and will not be repeated here.

[0097] The light source board 7 of the immune detection module in the photoelectric detection unit is an array of 6 patch LEDs with a spacing of 4mm. The center wavelength of the LED emitted light is 480nm; the center wavelength of the first filter 6 is 480nm and the bandwidth is 30nm; the spectrometer 5 is a short-wave pass dichroic mirror with a 50% splitting wavelength of 500nm, a transmittance of more than 90% for wavelengths below 500nm, and a reflectivity of more than 90% for wavelengths above 550nm; therefore, the light emitted by the light source board 7 is irradiated to the immune detection area of ​​the detection card 1 through the spectrometer 5; the signal light emitted by the immune detection area has a wavelength of 600nm, and the signal light is reflected by the spectrometer 5 and enters the second filter 4. The center wavelength of the second filter 4 is 600nm, the transmission spectrum range is 50nm, and the transmittance for wavelengths below 500nm is less than 0.01%.

[0098] The total white blood cell count in the photoelectric detection unit is detected using fluorescent staining. After fluorescent staining, the white blood cells in the blood sample have an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Therefore, the illumination light source 13 is an LED light source with a center wavelength of 480 nm, and the third filter 12 has a center wavelength of 480 nm and a bandwidth of 30 nm. Under the excitation of the light source, the white blood cells in the total white blood cell count detection area, dyed with fluorescent dye (cell fluorescent dye and surfactant), emit fluorescence. The fluorescence signal is reflected by the reflector 8 and imaged onto the second image sensor 11 through the second imaging lens 9 and the fourth filter 10. The fourth filter 10 is a filter with a center wavelength of 540 nm and a transmittance of less than 0.01% for light with a wavelength less than 500 nm.

[0099] Example 2: Immunoassay combined with leukocyte count

[0100] This embodiment uses the simultaneous detection device for immunoassay combined with cell counting provided in Example 1 to perform immunoassay combined with white blood cell counting, specifically comprising the following steps:

[0101] 1) Sample production

[0102] A 100 μL blood sample is taken using a sampling device such as a pipette and added to the total sample loading port 1c of the test card 1, or to the first sample loading port 216 and the second sample loading port 217. A portion of the blood sample (10 μL) automatically flows into the test cavity at the total white blood cell count detection area 1d, where it undergoes a hemolysis and staining reaction with the reagents (SYTO9 dye and quaternary ammonium salt in this embodiment) pre-encapsulated within the test cavity 22 of the total white blood cell count detection sheet 400. The remaining blood sample flows into the immune reaction area and undergoes an immune reaction with the immune targets there (in this embodiment, the conjugate pad 312 is immobilized with a quantum dot-labeled CRP monoclonal antibody and a quantum dot-labeled rabbit IgG; the detection strip 316 is coated with another CRP monoclonal antibody with a different epitope from the quantum dot-labeled CRP monoclonal antibody; and the quality control strip 317 is coated with anti-rabbit IgG). The reaction process takes about 10 minutes.

[0103] 2) Detection and imaging

[0104] After the sample has been placed for 10 minutes, the reaction is complete. Insert the test card 1 into the test device through the card insertion port 109, so that the total white blood cell count detection area 1d corresponds to the total white blood cell count detection module, and the immune detection area 1b corresponds to the immune detection module. At this time, click the test button on the display screen (touch screen) to start the test process.

[0105] The main control board 101 drives the excitation light source 13 of the total white blood cell count detection module to light up. Under the excitation of the light source, the stained white blood cells in the total white blood cell count detection area of ​​the detection card 1 emit fluorescence. The fluorescence signal is reflected by the reflector 8 and imaged by the second imaging lens 9 and the fourth filter 10 to the second image sensor 11. The collected white blood cell detection image is as follows: Figure 10 As shown, turn off the light source 13. After that, the main control board 101 drives the light source board 7 of the immune detection module to emit illumination light, which irradiates the immune detection area of ​​the detection card 1. The fluorescence emitted by the immune detection area of ​​the detection card 1 is reflected by the spectroscope 5, and then focused and imaged in the first image sensor 2 through the second filter 4 and the first imaging lens 3. The collected immune detection image is shown as follows: Figure 11 Turn off the light source as shown.

[0106] 3) Image processing

[0107] The main control board 101 transmits the collected total white blood cell count image and immune detection image data to the data processing unit for processing. For the total white blood cell count image processing, the magnification of the imaging optical path is pre-calibrated using a graticule. The number of fluorescent bright spots within the sample detection range is then calculated based on the image. The area of ​​the test cavity of the total white blood cell count detection strip corresponding to the detection range is then calculated based on the magnification. The solution volume corresponding to the detection range is then calculated based on the cavity thickness of the test cavity of the total white blood cell count detection strip, thereby obtaining the white blood cell concentration. The calculation formula is as follows:

[0108]

[0109] in: ILNum is the final total leukocyte concentration; N is the number of fluorescent bright spots; is the dilution ratio of the blood sample; S is the chamber area of ​​the test cavity of the total white blood cell count detection piece corresponding to the detection range, in mm 2 ; d is the chamber thickness, unit: μm;

[0110] For the processing of the immunoassay image: the data processing unit identifies and calculates the total fluorescence intensity of the fluorescent bright band in the image, and calculates the concentration of the analyte in the blood sample based on the fluorescence intensity.

[0111] 4) Result output

[0112] The test results are displayed on the display screen of the human-computer interaction unit, and users can also transmit and print the test results as needed.

[0113] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A synchronous detection device for immune detection combined with white blood cell count, characterized in that: include: case; a main control board, which is disposed in the housing; a photoelectric detection unit, which is disposed in the housing and electrically connected to the main control board, and includes an immune detection module and a total white blood cell count detection module, each detection module including an image sensor for capturing images; a data processing unit, integrated into the main control board, for processing and analyzing images captured by the image sensor; A human-computer interaction unit, electrically connected to the main control board; and A test card, which can be installed in a card insertion port provided on the housing for installing the test card, includes an immune detection sheet and a total white blood cell count detection sheet, which are respectively compatible with the immune detection module and the total white blood cell count detection module; The test card is a combined immune test and white blood cell count test card, including: A detection card bottom shell (200), a detection card upper cover (200'), and an immune detection piece (300) and a total white blood cell count detection piece (400) installed in a box body formed by the detection card upper cover and the detection card bottom shell, wherein: The detection card upper cover (200') is provided with a first sample loading hole (216) for loading a sample on the immune detection sheet (300), a second sample loading hole (217) for loading a sample on the total white blood cell count detection sheet (400), and a first interpretation window (218) for transmitting the immune detection area of ​​the immune detection sheet (300) and a second interpretation window (219) for transmitting the white blood cell detection area of ​​the total white blood cell count detection sheet (400). The detection card bottom shell (200) is provided with a third interpretation window (223) for transmitting the white blood cell detection area of ​​the total white blood cell count detection sheet (400). The second interpretation window (219) and the third interpretation window (223) are aligned. The first sample loading hole (216) and the second sample loading hole (217) are separated from each other, or connected to form a total sample loading port (1c). The total white blood cell count detection sheet (400) includes an assembly handle (21) and a test cavity (22) provided on the assembly handle. The test cavity (22) is a semi-open cavity formed by two parallel cavity side walls (23) with a certain gap, and has a test area (24), an injection port (25), and a drainage area (26) connecting the injection port (25) and the test area (24). The thickness of the test area (24) is Less than the thickness of the drainage area (26) The injection port (25) is aligned with the second sample addition hole (217), and the test area (24) is aligned with the second interpretation window (219) and the third interpretation window (223); The test cavity (22) is provided with at least one exhaust hole, which penetrates one cavity side wall of the test area (24) or the drainage area (26) or symmetrically penetrates two cavity side walls of the test area (24) or the drainage area (26); The exhaust hole is a symmetrical inverted cone-shaped through hole; The test area includes a test area with large thickness and a test area with small thickness; The injection port (25) is in a concave arc shape, and the angle α between the tangent of the downward arc and the horizontal reference plane of the injection port (25) is in the range of 15°-45°; The inner edge of the end edge of the cavity side wall (23) is provided with a transition fillet (210), and the transition fillet value R ranges from 0.2 mm to 1.5 mm.

2. The device according to claim 1, characterized in that The detection card bottom shell (200) and / or the detection card upper cover (200') are provided with at least one communication hole penetrating the box body, and the exhaust hole is connected to the outside atmosphere through the communication holes that match the number and position of the exhaust hole.

3. The device according to claim 1 or 2, characterized in that The injection port (25) is located at the upper edge opening of the two cavity side walls (23) of the test cavity (22), wherein the upper edge of one of the two cavity side walls (23) located at the injection port (25) is provided with an injection notch (28) so that the liquid sample can be injected through the injection notch (28).

4. The device according to claim 1, characterized in that The immunoassay sheet comprises: an analysis membrane, a binding pad, a sample pad, and a water-absorbing pad which are overlapped and fixed on a sticky backing. A detection band and a quality control band are arranged on the analysis membrane, and the detection band and the quality control band constitute the immunoassay area of ​​the immunoassay sheet.

5. The device according to claim 1, characterized in that The immune detection module (104) includes: A light source plate (7) for emitting light; a first optical filter (6) for transmitting light of a fixed wavelength range in the light emitted by the light source; A spectroscope (5) for transmitting light emitted by the light source of the first filter and reflecting fluorescence emitted by the immunoassay sheet; A second filter (4) is used to transmit light of a fixed wavelength range in the fluorescence emitted by the immunodetection piece reflected by the spectroscope; A first imaging lens (3) is used to image the fluorescence emitted by the immunodetection sheet; and A first image sensor (2) for focusing fluorescence imaging and transmitting the image to a data processing unit; The first filter is arranged between the spectroscope and the light source, the spectroscope is arranged between the immunodetection plate and the first filter, the imaging lens is arranged at a position where it can receive the fluorescence reflected by the spectroscope, and the imaging lens is arranged between the second filter and the first image sensor; The light source panel is composed of a plurality of LED light sources arranged in parallel.

6. The device according to claim 1, characterized in that The total white blood cell count detection module (105) includes: a light source (13) for emitting light; a third optical filter (12) for transmitting light of a fixed wavelength range in the light emitted by the light source; A reflector (8) is used to reflect the fluorescence emitted by the total white blood cell count detection sheet onto the imaging lens; A second imaging lens (9) is used to image the fluorescence emitted by the total white blood cell count detection sheet; A fourth filter (10) for transmitting light of a fixed wavelength range in the imaging fluorescence; and a second image sensor (11), for focusing cell fluorescence imaging and transmitting the imaging to a data processing unit; The third filter is arranged between the total white blood cell count detection plate and the light source, the total white blood cell count detection plate is arranged between the third filter and the reflector, the second imaging lens is arranged at a position where it can receive the fluorescence reflected by the reflector, and the fourth filter is arranged between the second image sensor and the second imaging lens.

7. A method for performing immunoassay combined with leukocyte counting using the device of claim 1, comprising the following steps: 1) Sample production Take a blood sample and add it to the total sample port of the test card or add it to the first sample well and the second sample well respectively, so that the blood sample flows into and fills the test cavity of the total white blood cell count detection piece and reacts with the pre-packaged reagent in the test cavity to form a white blood cell detection surface; The blood sample is made to flow to the reaction area of ​​the immune detection piece, and immune reaction occurs with the immune target in the immune reaction area to form an immune detection area; 2) Imaging detection Insert the test card containing the reacted sample obtained in step 1) into the card insertion port of the device, start the detection program to perform imaging detection, and use the image sensors of the immune detection module and the leukocyte detection module to respectively capture the fluorescence images of the samples in the corresponding detection areas; 3) Image processing The data processing unit processes the fluorescence images collected by the image sensor separately to obtain the results of immune detection and white blood cell detection; 4) Result output The detection results after image processing are displayed and output in the manual interaction unit and / or printed out through a printer.

8. The method according to claim 7, characterized in that The specific steps of the imaging detection in step 2) include: S1: The main control board drives the light source of the total white blood cell count detection module to light up. Under the excitation of the light source, the stained white blood cells in the total white blood cell count detection area emit fluorescence. The fluorescence signal is reflected by the reflector, and is imaged by the second imaging lens and the fourth filter to the second image sensor to obtain the total white blood cell count detection image. The light source is then turned off. S2: The main control board drives the light source board of the immunoassay module to light up. Under the excitation of the light source, the fluorescence emitted by the immunoassay area is reflected by the spectroscope, and then focused by the second filter and the first imaging lens in the first image sensor to obtain an immunoassay image. The light source is then turned off. S1 and S2 are performed synchronously or sequentially.

9. The method according to claim 7 or 8, characterized in that The specific method of image processing in step 3) is: For the processing of total white blood cell count detection images: Before processing, the magnification of the imaging light path is pre-calibrated with a graticule; then, based on the image, the number of fluorescent bright spots within the sample detection range is calculated. Furthermore, the cavity area of ​​the test cavity of the total white blood cell count detection strip corresponding to the detection range is calculated based on the magnification. Furthermore, based on the cavity thickness of the test cavity of the total white blood cell count detection strip, the solution volume corresponding to the detection range is calculated, and the white blood cell concentration is obtained. The calculation formula is as follows: in: ILNum is the final total leukocyte concentration; N is the number of fluorescent bright spots; is the dilution ratio of the blood sample; S is the chamber area of ​​the test cavity of the total white blood cell count detection piece corresponding to the detection range, in mm 2 ; d is the chamber thickness, unit: μm; For the processing of the immunoassay image: the data processing unit identifies and calculates the total fluorescence intensity of the fluorescent bright band in the image, and calculates the concentration of the analyte in the blood sample based on the fluorescence intensity.

Images