A white blood cell counting and typing instrument and a white blood cell counting and typing method

Abstract

The present invention discloses a white blood cell counting and typing instrument and a white blood cell counting and typing method, comprising: a human-computer interaction module, a photoelectric detection module, a main control unit and a detection card. The detection card is placed in the photoelectric detection module, and the main control unit is electrically connected to the human-computer interaction module and the photoelectric detection module. The white blood cell counting and typing instrument of the present invention does not require hemolysis or pH adjustment, has a short staining time, is simple to operate, and has accurate white blood cell classification. The white blood cell counting and typing method provided by the present invention can be used not only for the typing and counting of white blood cells, but also for the imaging counting of neutrophils, lymphocytes and monocytes.

Description

Technical Field

[0001] The present invention relates to the technical field of blood analysis and detection, and particularly to a white blood cell counting and typing instrument and a white blood cell counting and typing method. Background Art

[0002] Blood cells in the human body are divided into three categories: white blood cells, red blood cells, and platelets. White blood cells mainly include three categories: granulocytes, lymphocytes, and monocytes. Among them, granulocytes account for 50-70% of white blood cells, lymphocytes account for 20-40%, and monocytes account for 1-7%. Under normal circumstances, the total number of white blood cells in the human body and the percentage of each type of white blood cell are relatively stable, while inflammation or other diseases will cause changes in their values. That is to say, the occurrence of inflammation or other diseases in the body can cause changes in the total number of white blood cells and the percentage of each type of white blood cell. Therefore, the change in its value can be used as an indicator of human inflammation. By these value changes, it can be judged whether the body has inflammation or certain diseases. Therefore, classifying and counting white blood cells provides important indicators for modern clinical detection and diagnosis.

[0003] Currently, the methods for classifying and counting white blood cells mainly include the manual microscope method and the blood cell analyzer method.

[0004] The manual microscope method is to first hemolyze, that is, to destroy the red blood cells in the blood, then drop it into the counting tray, count the number of white blood cells within a certain range under the microscope, and finally convert it into the number of white blood cells per liter of blood. This method is manually detected and requires professional personnel to complete. It takes a long time and is not suitable for screening a large number of healthy people. In addition, the analysis results may also vary due to changes in operators, with large errors.

[0005] There are also the Coulter method and the flow cytometry method for determining the number of white blood cells. The counting principle of the Coulter method is based on the non-conductive properties of blood cells, using the resistance change caused by blood cells suspended in an electrolyte solution when passing through the counting aperture as the detection parameter for blood cell counting. Since the resistance change is related to the cell size, and different types of white blood cells have different volume sizes, different types of white blood cells can be counted accordingly. The flow cytometry method simultaneously uses principles such as photoelectric colorimetry, laser scattering, fluorescence staining technology, and sheath flow technology to detect the scattered light and fluorescence pulse signals generated by each cell one by one in the flow path, and then receives the scattered light and fluorescence pulse signals one by one. By calculating the number of received pulse signals, the classification and counting of different types of blood cells are achieved. The blood cell analyzer based on the flow cytometry method is currently the most mainstream inspection equipment, with the characteristics of fast detection speed, high efficiency, and the ability to avoid human factor interference. However, it has a high price, large volume, and high maintenance requirements, and is suitable for clinical tests in large hospitals, but it is difficult to be applicable to bedside detection, primary medical treatment, and the treatment of the wounded and sick in special environments.

[0006] In addition, there is now a new type of white blood cell counting device - HemoCue, which uses the principle of hemolysis counting method: first, the red blood cells in the blood are dissolved by a hemolytic agent, and then the white blood cells are stained with a cell staining agent (such as methylene blue, methyl green or gentian violet, etc.) to make the white blood cells colored. The stained sample is placed under transmitted light. Since the white blood cells become darker in color after staining, they will appear as dark spots on the image under the illumination of the transmitted light. The total number of white blood cells can be calculated by counting the number of dark spots, and different types of white blood cells can be calculated based on the morphology of the dark spots. However, the hemolysis counting method used by HemoCue has the following disadvantages: First, this method uses the transmission absorption method and is easily interfered by impurities; second, the detection time window is short and the detection must be strictly controlled within 2 - 10 minutes. If the time is too short, the red blood cells are not completely dissolved, and if the time is too long, some white blood cells will also be dissolved; third, the accuracy of distinguishing white blood cell types by geometric features is low. Summary of the Invention

[0007] The object of the present invention is to address the technical defects existing in the prior art. In the first aspect, a white blood cell counting and typing instrument for rapidly detecting the number and type of blood white blood cells is provided, including:

[0008] A human - machine interaction module, which is used for inputting user instructions and outputting detection results;

[0009] An optoelectronic detection module, which is used for detecting and receiving the optical signals emitted by a blood sample stained with a nucleic acid staining agent;

[0010] A main control unit, which is used for transmitting the user instructions input by the human - machine interaction module to the optoelectronic detection module, controlling the optoelectronic detection module to perform detection, processing and calculating the optical signals received by the optoelectronic detection module to complete the counting and typing of white blood cells, and transmitting the detection results of the optoelectronic detection module to the human - machine interaction module; and

[0011] A detection card, which is at least used for carrying a blood sample, is provided with a hollow chamber, and one or more detection areas are arranged in the chamber;

[0012] The detection card is placed in the optoelectronic detection module, and the detection area is located in the imaging area of the optoelectronic detection module; the main control unit is electrically connected to the human - machine interaction module and the optoelectronic detection module.

[0013] The optoelectronic detection module includes

[0014] A light source, which is used for emitting fluorescence;

[0015] A collimating lens, which is used for refracting the light emitted by the light source into parallel light;

[0016] A filter, which is used for transmitting the light within a fixed wavelength range in the light emitted by the light source;

[0017] A fluorescence filter for transmitting light within a fixed wavelength range in the fluorescence excited by the test card;

[0018] An imaging lens for imaging the light transmitted through the fluorescence filter; and

[0019] An image sensor for receiving the image formed by the imaging lens;

[0020] The test card is placed at a position where the transmitted light of the filter can directly shine, the fluorescence filter is placed at a position where it can receive the fluorescence excited by the test card, and both the light source and the image sensor are electrically connected to the main control unit.

[0021] In the photoelectric detection module, the light source, the collimating lens, the filter, the fluorescence filter, the imaging lens and the image sensor are arranged at intervals and in parallel in sequence. The test card is located between the filter and the fluorescence filter and is parallel to the filter. The light emitted by the light source sequentially passes through the collimating lens, the filter, the test card, the fluorescence filter, the imaging lens and the image sensor.

[0022] The photoelectric detection module further includes a beam splitter for reflecting the transmitted light of the filter to the detection area of the test card and simultaneously transmitting the excited fluorescence of the detection area to the fluorescence filter;

[0023] Preferably, the light source, the collimating lens, the filter and the beam splitter are arranged at intervals in sequence. The collimating lens and the filter are parallel, and the included angle between the beam splitter and the filter is 45°. The test card, the beam splitter, the fluorescence filter, the imaging lens and the image sensor are arranged at intervals in sequence. The test card, the fluorescence filter, the imaging lens and the image sensor are parallel to each other. The included angle between the test card and the filter is 90°, and the included angle between the beam splitter and the test card is 45°. The light emitted by the light source sequentially passes through the collimating lens, the filter, the beam splitter, the test card, the beam splitter, the fluorescence filter, the imaging lens and the image sensor.

[0024] The photoelectric detection module further includes a scattered light imaging block for distinguishing lymphocytes and monocytes in white blood cells;

[0025] Preferably, the scattered light imaging block includes a scattered light illumination light source, a scattered light lens and a scattered light filter which are arranged at intervals and in parallel in sequence. The detection area of the test card is placed at a position where the transmitted light of the scattered light filter can irradiate. The light emitted by the scattered light illumination light source sequentially passes through the scattered light lens and the scattered light filter and irradiates on the detection area of the test card. The scattered light illumination light source is electrically connected to the main control unit.

[0026] An anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the hollow chamber of the test card; preferably, the anticoagulant is one or more of ethylenediaminetetraacetate, citrate, oxalate, heparin, etc.; the hemolytic agent is selected from surfactants such as tritonX-100, quaternary ammonium salts or saponin, and the staining agent is selected from acridine orange fluorescent dyes.

[0027] The hollow chamber in the detection card is a sampling and detection chamber, which is a semi-open cavity formed by two parallel chamber side walls with a certain gap. It has a detection area, a sampling port, and a diversion groove area connecting the sampling port and the detection area. The thickness H of the detection area 检 is less than the thickness H of the diversion groove area 导 ;

[0028] Preferably, the sampling port is in a concave arc shape, and the included angle α between the tangent of its downward sliding arc and the horizontal reference plane of the sampling port determines the flow direction of the liquid sample to be tested into the diversion groove area. The value range of the included angle α is 15° to 45°.

[0029] The sampling port is located at the upper edge opening of the two chamber side walls of the sampling and detection chamber. There is a sampling notch at the upper edge of one of the chamber side walls at the sampling port.

[0030] There are two or more detection areas. The thicknesses of the detection areas are not equal, and they are spatially independent but connected. The one with a larger thickness is used for cell counting, and the one with a smaller thickness is used for cell typing.

[0031] In a second aspect, the present invention provides a method for counting and typing white blood cells for quickly detecting the number and type of white blood cells. Using the above-mentioned white blood cell counting and typing instrument, it sequentially includes steps such as diluting whole blood into a sample liquid to be tested, adding the sample liquid to be tested into the detection card for hemolysis and fluorescence staining, putting the detection card into the white blood cell counting and typing instrument, fluorescence excitation and detection, image processing and calculation, etc.;

[0032] Preferably, the fluorescence excitation and detection are specifically three methods:

[0033] Method 1: The main control unit controls the light source to turn on. The light emitted by the light source sequentially passes through a collimating lens, a filter, the detection card, a fluorescence filter, an imaging lens, and an image sensor. The image sensor transmits the collected fluorescence image to the main control unit;

[0034] Method 2: The main control unit controls the light source to turn on. The light emitted by the light source sequentially passes through a collimating lens, a filter, a beam splitter, the detection card, a beam splitter, a fluorescence filter, an imaging lens, and an image sensor. The image sensor transmits the collected fluorescence image to the main control unit;

[0035] Method 3: The main control unit first controls the light source to turn on and turns off the scattered light illumination light source. The light emitted by the light source sequentially passes through a collimating lens, a filter, a detection card, a fluorescence filter, an imaging lens, and an image sensor. The image sensor transmits the collected fluorescence image to the main control unit. Then, the main control unit controls the scattered light illumination light source to turn on and turns off the light source. The light emitted by the scattered light illumination light source sequentially passes through a scattered light lens, a scattered light filter, a detection card, a fluorescence filter, an imaging lens, and an image sensor. The image sensor transmits the collected scattered light image to the main control unit.

[0036] The image processing and calculation are specifically as follows: The main control unit first counts all fluorescence points and uses the number of independent fluorescence points as the total number of white blood cells. Then, it separately counts the green light intensity and red light intensity of all fluorescence points. Those with a higher proportion of red light intensity are regarded as the number of granulocytes in white blood cells, and those with a higher proportion of green light intensity are regarded as the number of lymphocytes and monocytes.

[0037] Preferably, the scattered light signal is finally processed. Those with a relatively weak scattered light signal are regarded as the number of lymphocytes, and those with a relatively strong scattered light signal are regarded as the number of monocytes.

[0038] The specific operation of adding the sample liquid to be tested into the detection card is as follows: Dip the sampling port into the sample liquid to be tested for sampling, and make the sample liquid to be tested flow into the detection area from the diversion groove area under the action of capillary force and fill it. Among them, the capillary force satisfies the following relationship with the thickness of the detection area and the thickness of the diversion groove area:

[0039]

[0040] Or,

[0041] Inject the sample liquid to be tested into the sampling and detection cavity through the sampling notch for sampling, and make the sample liquid to be tested flow into the detection area from the diversion groove area and fill it.

[0042] In the white blood cell counting and typing instrument of the present invention, the thickness of the reaction cavity is 30 - 100 μm, which can make the overlapping layer number of white blood cells less than 5, avoid the problem that the fluorescence irradiating the lower-layer white blood cells cannot pass through due to cell overlap, and improve the accuracy of the detection result. In the white blood cell counting and typing instrument of the present invention, the dye is stored in the state of dry powder, which is convenient for storage and transportation.

[0043] The white blood cell counting and typing instrument of the present invention is easy to use. Just add fresh blood into the reaction cavity. The blood does not need to be diluted or centrifuged. After adding the blood, wait for 3 minutes for measurement. And the blood can be either fingertip blood or venous blood. The white blood cell counting and typing instrument of the present invention can be used in cooperation with a slit microfluidic chip based on slit structure liquid diversion to achieve the purpose of rapid, automatic, and accurate measurement of trace liquid, and complete the typing and counting of white blood cells in liquid samples at one time.

[0044] The white blood cell counting and typing method of the present invention stains white blood cells with a fluorescent dye that stains nucleic acids. Under fluorescence excitation, red blood cells and plasma are not affected by the staining agent and have no fluorescence; granulocytes combine with the staining agent to emit orange-yellow light, and lymphocytes and monocytes emit bright green light. Granulocytes and lymphomonocytes in white blood cells can be classified according to the emitted fluorescence color. Since 90% of lymphocytes in the blood have a diameter of 5-8 μm and monocytes have a diameter of 10-20 μm, lymphocytes and monocytes can be further classified according to cell diameter. The method of the present invention does not require hemolysis or pH adjustment, has a short staining time, is simple to operate, and accurately classifies white blood cells. The white blood cell counting and typing method provided by the present invention can be used not only for the typing and counting of white blood cells, but also for the imaging and counting of neutrophils, lymphocytes, and monocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The schematic diagram of the principle of the white blood cell counting and typing instrument in Embodiment 1 is shown;

[0046] Figure 2 The schematic diagram of the principle of the white blood cell counting and typing instrument in Embodiment 2 is shown;

[0047] Figure 3 The schematic diagram of the principle of the white blood cell counting and typing instrument in Embodiment 3 is shown;

[0048] Figure 4 The structural schematic diagram of the white blood cell counting and typing instrument of the present invention is shown.

[0049] Figure 5 The flow chart of the white blood cell counting and typing method of the present invention is shown;

[0050] Figure 6 The photo of white blood cells after fluorescence staining by the white blood cell counting and typing method of the present invention is shown;

[0051] Figure 7 The red and green fluorescence scatter plots obtained by fluorescence image processing of the present invention are shown;

[0052] Figure 8 The fluorescence photo of counting and typing white blood cells by the method of the present invention is shown;

[0053] Figure 9 The scattered light photo of counting and typing white blood cells by the method of the present invention is shown;

[0054] Figure 10 The histogram of scattered light image processing of lymphocytes and monocytes by the method of the present invention is shown;

[0055] Figures 11A - 11C The structural schematic diagram of the detection card in Embodiment 1 of the present invention is shown;

[0056] Figure 12A It is a schematic plan view of the detection card according to the fourth embodiment of the present invention;

[0057] Figure 12B It is a schematic three-dimensional structure view of the detection card according to the fourth embodiment of the present invention;

[0058] Figure 12C is Figure 12A The sectional view taken along the line C1-C1 in Detailed implementation manners

[0059] Since red blood cells and plasma in blood have no nucleic acid, while white blood cells have cell nuclei, the present invention utilizes this difference to type and count white blood cells, that is, staining the cells in blood with a fluorescent dye for staining nucleic acid (such as acridine orange, SYTO9, etc.). Red blood cells and plasma have no nucleic acid, so there is no fluorescence; platelets also do not produce fluorescence; white blood cells have cell nuclei and can emit fluorescence. With this principle, red blood cells, plasma, and white blood cells can be distinguished, and the total number of white blood cells can be calculated according to one cell corresponding to each fluorescent dot. Further, since granulocytes emit orange-yellow fluorescence after staining, while lymphocytes and monocytes emit bright green fluorescence after staining, this principle can be used to distinguish granulocytes from lymphocytes and monocytes, and the number of granulocytes can be calculated according to one cell corresponding to each fluorescent dot. Finally, taking advantage of the different diameters of lymphocytes and monocytes, these two types of cells are distinguished by the principle of scattered light, and the numbers of lymphocytes and monocytes are calculated, while completing the counting and typing of white blood cells.

[0060] On this basis, the present invention provides a white blood cell counting and typing instrument, as Figure 4 shown, mainly including a human-computer interaction module II, a main control unit 8, a photoelectric detection module I, and a detection card 4.

[0061] The detection card 4 is at least used to carry a blood sample, and it has a hollow chamber. The present invention also specially designs that anticoagulant, hemolytic agent, and staining agent are pre-encapsulated on the inner wall of this chamber; the anticoagulant is one or more of ethylenediaminetetraacetate, citrate, oxalate, heparin, etc.; the hemolytic agent is selected from surfactants such as tritonX-100, quaternary ammonium salt, or saponin, and the staining agent is selected from acridine orange fluorescent dye. The detection card 4 includes a support substrate and a hollow chamber provided on the support substrate. This chamber is a sampling and detection chamber, which is a semi-open cavity formed by two parallel chamber side walls with a certain gap, and has one or more detection areas with different thicknesses, a sampling port, and a diversion groove area connecting the sampling port and the detection area. The sampling port is in a concave arc shape, and the included angle α between the tangent of its downward sliding arc and the horizontal reference plane of the sampling port determines the flow direction of the liquid sample to be detected into the diversion groove area, and the value range of the included angle α is 15° to 45°; an optimized detection card also has a sampling notch in the sampling port to facilitate pipetting and sample injection.

[0062] The human-computer interaction module II mainly includes the housing 14 of the instrument, the detection card compartment door, the display screen 11 and the function keys. The housing 14 is used to protect the internal components of the instrument. The detection card compartment door is used to insert the detection card 4. The display screen 11 and the function keys complete the input of user instructions and the output of detection results. The function keys include the power on / off key, the detection key and the return key. The power on / off key is used to control the startup and shutdown of the instrument. The detection key is used to start the detection program. The return key is used to return to the main page.

[0063] The main control unit 8 is responsible for controlling the working process of the instrument. It is a 4412-type core control board purchased from Guangzhou Friendly Electronic Technology Co., Ltd., which is electrically connected to the function keys and the display screen on the human-computer interaction module and is also electrically connected to the optoelectronic detection module. After the human-computer interaction module sends an instruction to the main control unit 8, the main control unit 8 controls the turning on or off of the light source in the optoelectronic detection module, the acquisition of sample images, the image processing of the acquired sample images, and converts the digital signal of sample typing and counting into a readable result and feeds it back to the display screen of the human-computer interaction module.

[0064] Optical and electrical detection module I includes a light source 1, a collimating lens 2, a filter 3, a fluorescence filter 5, an imaging lens 6, and an image sensor 7. A detection card 4 is provided between the filter 3 and the fluorescence filter 5. The main control unit is electrically connected to the light source 1 and the image sensor 7. The light source 1 is a fluorescence excitation light source, and the wavelength of the emitted light beam is determined by the fluorescence characteristics of the sample to be measured. For example, for SYTO-9 stained cells, the wavelength of the light source 1 is 470 - 490 nm. The light source 1 is placed at the rear focal point of the collimating lens 2 (with the light propagation direction as the front), the focal length of the collimating lens 2 is 10 - 20 mm, and the light emitted by the light source 1 forms a parallel light beam after passing through the collimating lens 2. The light beam passing through the collimating lens 2 is filtered by the filter 3 and then irradiated on the detection card 4. The filter 3 is a band-pass filter, and its transmission wavelength is determined by the fluorescence characteristics of the sample to be measured. For example, for SYTO-9 stained cells, the transmission wavelength of the filter 3 is 470 - 490 nm. The target cells in the detection card 4 emit fluorescence under the excitation of the light beam irradiation. The fluorescence is successively focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6. The transmission wavelength range of the fluorescence filter 5 is determined by the fluorescence characteristics of the sample to be measured. For example, for SYTO-9 stained cells, the transmission wavelength range of the fluorescence filter 5 is 520 - 650 nm. In addition, the fluorescence filter 5 blocks the light transmitted by the filter 3, and the blocking efficiency is better than 0.01%, that is, the transmittance of the light transmitted through the filter 3 is less than 0.01% when passing through the fluorescence filter 5. This is because the fluorescence wavelength emitted by the sample after being excited by light is 520 - 650 nm, and the fluorescence filter 5 filters out the light outside this range to avoid the interference of the light beam before excitation. The object-side numerical aperture of the imaging lens 6 is greater than 0.1, and the object-image magnification is not less than 0.5X. The image sensor 7 is a area array camera, and the resolution is greater than 1 million pixels. Both the light source 1 and the image sensor 7 are connected to the main control unit 8. The main control unit 8 controls the on or off of the light source 1 and the image acquisition of the image sensor 7. After the main control unit 8 obtains the image signal of the image sensor 7, it further completes image processing, calculates the characteristics and quantity of the cells in the image, respectively obtains the total number of white blood cells, that is, the quantity of granulocytes, and the combined quantity of lymphocytes and monocytes, and then transmits them to the human-computer interaction module for output through the display screen.

[0065] Furthermore, the optical and electrical detection module may also include a scattered light imaging block. Since the light intensity of scattered light is sensitive to cell size, different-sized cells can be distinguished by scattered light. Since the fluorescence of both lymphocytes and monocytes is green, but the volume of monocytes is larger than that of lymphocytes, after adding the scattered light imaging block, lymphocytes and monocytes can be further distinguished by scattered light. The scattered light imaging block includes a scattered light illumination light source 1', a scattered light lens 2', and a scattered light filter 3' arranged in sequence.

[0066] The scattered light illumination source 1' is a scattered light imaging illumination source, preferably a light source with a wavelength greater than 600nm, located at the rear focus of the scattered light lens 2' (with the direction of light propagation as the front), and a scattered light filter 3' is provided in front of the scattered light lens 2' to filter out light with a wavelength less than 600nm. The light emitted by the scattered light illumination source 1' is converged by the scattered light lens 2' to form parallel light, and the parallel light is filtered out by the scattered light filter 3' and irradiated on the detection card 4 after the stray light is filtered out. Lymphocytes and monocytes of different sizes will produce scattered light of different light intensities under the irradiation of scattered light. The scattered light passing through the scattered light filter 3' is irradiated on the detection card 4 at an incident angle greater than 45 degrees, so that only the scattered light irradiated on the detection card 4 can enter the imaging lens 6, while the direct transmitted light irradiated on the detection card 4 cannot enter the imaging lens 6. The scattered light reflected by the detection card 4 is focused and imaged in the image sensor 7 through the fluorescent filter 5 and the imaging lens 6 in turn.

[0067] The scattered light illumination source 1' is also connected to the main control unit 8, and the main control unit 8 controls the on or off of the scattered light illumination source 1'; the light source 1 and the light source 1' are lit in time-sharing manner and cannot be lit at the same time; after the main control unit 8 obtains the image signal of the image sensor 7, it first processes the fluorescence signal, calculates the number of granulocytes and the total number of lymphocytes + monocytes, and then processes the scattered light signal to calculate the number of lymphocytes and monocytes respectively, and then transmits it to the human-computer interaction module through the display screen for output.

[0068] The method for using the leukocyte counting and typing instrument of the present invention is as follows: when using the leukocyte counting and typing instrument, the user clicks the detection function button on the touch screen, and presses the detection card hatch 13 on the right side inward according to the instrument prompt, and the hatch will automatically unlock and pop out the sample bracket; the detection card is placed on the sample bracket, and then the detection card hatch is pushed in and closed, and the sample is added. At this time, the confirmation button on the touch screen is clicked to start the detection process.

[0069] The main control unit drives the light source to light up, and then controls the image sensor to collect the fluorescence image of the sample; after the fluorescence image is collected, the fluorescent light source is turned off, the scattered light illumination source is turned on, and the image sensor is synchronously controlled to collect the scattered image of the sample, and then the scattered light illumination source is turned off. After the fluorescence image and scattered image are collected, the main control unit performs image processing and displays the detection results on the touch screen.

[0070] The present invention also provides a method for counting and typing white blood cells, the process of which is as follows: Figure 5 As shown, the following steps are included:

[0071] (1) Sampling

[0072] Using whole blood as the test sample, mix the whole blood with a diluent (the diluent is pure water, physiological saline or phosphate buffer) at a ratio of 1:3 to prepare a test sample solution;

[0073] (2), Staining

[0074] Add the test sample solution to the test area of the test card 4 and let it stand for 2 - 3 minutes. The test sample solution dissolves the reagent in the test area of the test card and reacts, completing the hemolysis of red blood cells and fluorescence staining of white blood cells in the whole blood under the action of the reagent; the reagent in the test area of the test card includes an anticoagulant, a hemolytic agent and a staining agent. The anticoagulant is one or more of ethylenediaminetetraacetate, citrate, oxalate, heparin, etc.; the hemolytic agent is selected from surfactants such as tritonX - 100, quaternary ammonium salts or saponin, and the staining agent is selected from acridine orange fluorescent dye;

[0075] (3), Fluorescence excitation and detection

[0076] Insert the reacted test card 4 into the above-mentioned white blood cell counting and typing instrument, and place the test area of the test card in the imaging area of the photoelectric detection module; the user starts the detection process through key operation, the main control unit 8 drives to light up the illumination light source, and then controls the image sensor 7 to collect fluorescence images.

[0077] (4), Image processing and calculation

[0078] After the image acquisition is completed, the main control unit 8 performs image processing: first, count all the fluorescence points, and use the number of independent fluorescence points as the directly measured total number of white blood cells; then, for all the fluorescence points, count their green light intensity and red light intensity respectively, and calculate the number of granulocytes according to the proportion of the red light intensity, and calculate the number of lymphocytes and monocytes according to the proportion of the green light intensity.

[0079] The following combines specific embodiments to more specifically illustrate the content of the present invention and further elaborate on the present invention, but these embodiments are by no means a limitation to the present invention.

[0080] Example 1:

[0081] Figures 11A - 11C This is a structural example of the test card 4 of the present invention, which is designed in the form of a slit microfluidic chip. Figures 11A - 11CIn the first embodiment shown, the test card includes a support substrate 21 and a sampling and detection cavity 22 provided on the support substrate 21. Among them, the support substrate 21 is the hand-held part of the test card, and its shape is designed to be suitable for holding. For example, in the first embodiment, the hand-held part is rectangular, and the sampling and detection cavity 22 with an arc-shaped edge extends from its front end; the sampling and detection cavity 22 can be integrally formed with the support substrate 21, or the sampling and detection cavity 22 is bonded to the front end of the support substrate 21. The sampling and detection cavity 22 is a semi-open cavity formed by two chamber side walls 24 with a certain gap and parallel to each other, including a sampling port 27, a sampling notch 29, a detection area 25, and a diversion channel area 26 connecting the sampling port 27 and the detection area 25. Among them:

[0082] The sampling port 27 is located at the opening on the edge of the sampling and detection cavity 22, and the sample can be added by the active suction method through the sampling port 27.

[0083] The detection area 25 is located inside the sampling and detection cavity 22. The shape of the detection area 25 can be rectangular, square, trapezoidal, circular, or a combination of an arc and other shapes, and each shape can be provided with rounded corners, right angles, or a combination of rounded corners and right angles; there is at least one detection area 25, and it can have a single thickness H 检 , and the thickness range is generally 60 - 120 μm. As Figure 11B shown, when the sample enters the detection area 25, a detection surface is formed. For the detection area 25 with a large thickness, the sample carrying capacity per unit area of the detection surface is large and the depth of field is large, which is suitable for the overall accurate measurement of the number of cells; when the thickness is small, the spreading area of the same volume of liquid sample on the detection surface is large, which is suitable for the precise distinction of cell types.

[0084] The diversion channel area 26 is located inside the sampling and detection cavity 22 and is connected to the sampling port 27 and the detection area 25. The thickness range of the diversion channel area 26 is generally 120 μm to 500 μm. As Figure 11C shown, the thickness of the detection area 25 is less than the thickness of the diversion channel area 26, and the liquid sample enters from the sampling port 27 and is uniformly and quickly introduced and fills the entire sampling and detection cavity 22 through the flow path formed by the diversion channel area 26.

[0085] The thickness of the detection area 25 and the thickness of the diversion channel area 26 determine the flow state of the liquid sample to be tested in the diversion channel area 26 and the spreading state in the detection area 25. The liquid sample to be tested enters the diversion channel area 26 through the sampling port 27. The sample inhalation stage belongs to the pure inertial rising stage under the action of capillary force. According to the formula 1) of the pure inertial rising stage of capillary flow, the relationship between the volume of the liquid sample to be tested inhaled and the thickness of the detection area 25 can be obtained:

[0086]

[0087] In order to ensure that the liquid sample continuously flows from the guide groove area 26 into the detection area 5 under the action of capillary force and fills the sampling detection cavity 22, the capillary force is required to be greater than zero. 检 , the thickness of the guide groove area H 导 The following relationship exists:

[0088]

[0089] Figures 11A - 11C The detection area 25 shown is provided with a single thickness H located in the sampling detection cavity 22. 检 , its shape is a rounded rectangle. When the thickness H of the detection area 25 检 When the thickness is large, the detection surface formed on the chamber side wall 24 has a large depth of field and a large sample carrying capacity per unit area, which is suitable for the overall accurate measurement of the number of cells. The thickness of the detection area 25 is preferably 90 μm-120 μm; when the thickness H of the detection area 25 is 检 When the thickness is small, the depth of field of the detection surface formed on the chamber side wall 24 is small and the sample spreading area per unit volume is large, which is suitable for precise differentiation of cell types. The thickness of the detection area 25 is preferably 60 μm-90 μm.

[0090] Furthermore, a sampling notch 29 is provided at the upper edge of one of the two chamber side walls 24 at the sampling port 27, so that the sample can be added through the sampling notch 29 by injection. The sampling port 27 and the sampling notch 29 are compatible with both active injection and passive suction liquid sampling modes. The sampling port 27 is concavely arc-shaped, and the angle α between the tangent of its downward arc (the arc on the left in the figure) and the horizontal reference plane of the sampling port 27 (see Figure 11A ), the angle determines the direction of flow of the liquid sample to be tested into the guide groove area 26, and the preferred range of the angle α is 15° to 45°, which can ensure that the liquid sample to be tested spontaneously flows into and fills the sampling detection cavity 22 in a predetermined manner.

[0091] Specifically, the guide groove area 26 is located in the sampling and detection cavity 22, connecting the sampling port 27 and the detection area 25, and the thickness H of the guide groove area 26 is 导 Greater than the thickness H of the detection area 25 检 , try to avoid bubbles during the injection process; to ensure that the liquid sample to be tested can flow continuously from the guide groove area 26 into the detection area 5 under the action of capillary force and fill the sampling detection area 25, the capillary pressure is required to be greater than zero, and the capillary force is calculated according to formula 2).

[0092] The structural design of the first embodiment is suitable for cell counting and typing detection with a small number of cells per unit volume or low type abundance, or for applications where cell counting or cell typing is required. According to the application, a detection area 25 with a single thickness is selected, and a suitable thickness H is set. 检for high-precision measurement of cell numbers or high-precision analysis of individual cell types.

[0093] Of course, in this embodiment, a detection area 25 may also have multiple thicknesses H 检1 , H 检2 etc., and each thickness value of the detection area 25 is less than the thickness of the diversion groove area 26. The capillary force for driving the liquid sample to be tested into the sampling and detection cavity 22 and the thicknesses of each part of the detection area 25 and the thickness H of the diversion groove area 导 still satisfy Equation (2). When the sample enters the detection area 25 with thickness changes, detection surfaces with different depths of field and different spreading states can be formed. Data processing of cells in detection areas with different thicknesses can simultaneously take into account the precision measurement of cell counting and typing.

[0094] Embodiment 2:

[0095] The detection card of this embodiment is the same as that of Embodiment 1, and the staining agent is acridine orange.

[0096] The human-computer interaction module II mainly includes the housing 14 of the instrument, the detection card hatch, the display screen 11 and the function keys. The housing 14 is used to assemble the internal components of the instrument and install the display screen, function keys, etc. The detection card hatch is used to place the detection card 4. The display screen 11 and the function keys complete the input of user instructions and the output of detection results; the function keys include the power key, the detection key and the return key. The power key is used to control the start and stop of the instrument. The detection key is used to start the detection program. The return key is used to return to the main page.

[0097] The main control unit 8 is responsible for controlling the working process of the instrument, is electrically connected to the function keys and the display screen on the human-computer interaction module, and is electrically connected to the photoelectric detection module. After the human-computer interaction module issues an instruction to the main control unit 8, the main control unit 8 controls the on or off of the light source in the photoelectric detection module, the acquisition of the sample image, the image processing of the acquired sample image, and converts the digital signal of the sample typing and counting into a readable result and feeds it back to the display screen of the human-computer interaction module.

[0098] The photoelectric detection module I, as Figure 1 shown, sequentially includes a light source 1, a collimating lens 2, a filter 3, a fluorescence filter 5, an imaging lens 6, and an image sensor 7 according to the optical path direction, that is, the light source 1, the collimating lens 2, the filter 3, the fluorescence filter 5, the imaging lens 6, and the image sensor 7 are sequentially arranged on a straight line; a detection card 4 is provided between the filter 3 and the fluorescence filter 5; the main control unit is electrically connected to the light source 1 and the image sensor 7. Among them,

[0099] The light source 1 is a blue LED with a wavelength of 470 - 490 nm. The light source 1 is placed at the rear focal point of the collimating lens 2 (with the direction of light propagation being the front), and the light emitted by the light source 1 forms parallel light after passing through the collimating lens 2. The parallel light is then filtered by the filter 3 and irradiated on the test card 4.

[0100] The filter 3 is a band - pass filter with a transmission wavelength of 470 - 490 nm. The target cells in the test card 4 emit fluorescence under the excitation of the light beam. The fluorescence is successively focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6.

[0101] The transmission wavelength range of the fluorescence filter 5 is 520 - 650 nm; in addition, the fluorescence filter 5 blocks light with a wavelength of 450 - 500 nm (the transmission wavelength of the light passing through the filter 3 is within this range), and the cut - off efficiency is better than 0.01%. That is, the transmittance of light with a wavelength of 450 - 500 nm (the transmission light wavelength of the light passing through the filter 3 is within this range) passing through the fluorescence filter 5 is less than 0.01%. This is because the fluorescence wavelength emitted by the sample after being excited by light is 520 - 650 nm, and the fluorescence filter 5 is used to filter out light outside this range to avoid interference from the light beam before excitation.

[0102] The focal length of the collimating lens 2 is 10 - 20 mm. The object - side numerical aperture of the imaging lens 6 is greater than 0.1, and the object - image magnification is not less than 0.5X. The image sensor 7 is a area - array camera with a resolution of 3 million pixels.

[0103] Both the light source 1 and the image sensor 7 are connected to the main control unit 8. The main control unit 8 controls the turning on or off of the light source 1 and the image acquisition of the image sensor 7; after the main control unit 8 obtains the image signal of the image sensor 7, it further completes image processing, calculates the characteristics and quantity of the cells in the image, and then transmits them to the human - machine interaction module by the main control unit 8 for output through the display screen.

[0104] The counting and typing method of this embodiment includes the following steps:

[0105] (1). Sampling

[0106] Using whole blood as the test sample, mixing the whole blood with physiological saline at a ratio of 1:3 to prepare a sample solution to be tested;

[0107] (2). Staining

[0108] Adding the sample solution to be tested to the detection area of the test card 4 and standing for 2 - 3 minutes. The sample solution to be tested dissolves the reagent in the detection area of the test card and reacts, completing the hemolysis of red blood cells and the fluorescence staining of white blood cells in the whole blood under the action of the reagent. The anticoagulant is citrate, and the hemolytic agent is tritonX - 100 (polyethylene glycol octyl phenyl ether);

[0109] (3). Fluorescence excitation and detection

[0110] Insert the detected test card 4 after the reaction into the above-mentioned white blood cell counting and typing instrument, and place the detection area of the test card 4 in the imaging area of the photoelectric detection module; the user starts the detection process through key operations, the main control unit 8 drives to light up the illumination light source, and then controls the image sensor 7 to collect fluorescence images, as Figure 6 shown.

[0111] (4), Image processing and calculation

[0112] After the image acquisition is completed, the main control unit performs image processing. First, count all the fluorescence points, and use the number of independent fluorescence points as the total number of white blood cells N measured directly; then, for all the fluorescence points, count their green light intensity and red light intensity respectively, and calculate a two-dimensional scatter plot based on the green light intensity and red light intensity. Among them, the granulocytes with a high proportion of red light intensity are counted as N1, and the lymphocytes and monocytes with a high proportion of green light intensity are counted as N2, as Figure 7 shown, N = N1 + N2.

[0113] Example 3:

[0114] This example is only different from Example 2 in the photoelectric detection module, as Figure 2As shown in the figure, it consists of a light source 1, a collimating lens 2, a filter 3, a beam splitter 9, a fluorescence filter 5, an imaging lens 6, an image sensor 7, a control unit 8, etc. The light source 1, the collimating lens 2, the filter 3 and the beam splitter 9 are arranged in sequence on a horizontal straight line along the light propagation direction. The beam splitter 9, the fluorescence filter 5, the imaging lens 6 and the image sensor 7 are arranged in sequence on another vertical straight line. The horizontal straight line where the light source 1, the collimating lens 2 and the filter 3 are located is perpendicular to the vertical straight line where the fluorescence filter 5, the imaging lens 6, etc. are located. The focus (i.e., the foot of the perpendicular) of the two straight lines is the position where the beam splitter 9 is located. The angle between the beam splitter 9 and the horizontal straight line is 45°, and the angle between the beam splitter 9 and the vertical straight line is 45°. The test card 4 is located on the vertical straight line where the fluorescence filter 5, the imaging lens 6, etc. are located, and is located on both sides of the beam splitter 9 with the fluorescence filter 5 respectively, so that the beam splitter can reflect the light filtered by the filter 3 to the test card 4, and can also transmit the fluorescence emitted from the test card 4 to the fluorescence filter 5; the beam splitter reflects the light emitted by the light source 1 and transmits the fluorescence emitted by the test card 4, which can greatly prevent the light emitted by the light source 1 from directly entering the imaging lens 6 and reduce the interference to fluorescence imaging. The light source 1 is a blue LED with an emission light wavelength of 470 - 490 nm; the light source 1 is placed at the rear focus of the collimating lens 2, so that the light emitted by the light source 1 becomes parallel light after passing through the collimating lens 2, and the parallel light reaches the beam splitter 9 after being filtered by the filter 3. The filter 3 is a band-pass filter with a transmission wavelength of 470 - 490 nm. The beam splitter 9 is a dichroic beam splitter with a reflectivity greater than 90% for the light emitted by the light source 1 and a transmittance greater than 90% for the fluorescence emitted by the test card 4. Thus, the parallel light reaching the beam splitter 9 is reflected by the beam splitter and irradiates the test card 4. The target cells in the test card 4 emit fluorescence under the excitation of the light beam of the light source 1. After the fluorescence is transmitted by the beam splitter 4, it is then focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6 in sequence. The transmission wavelength range of the fluorescence filter 5 is 520 - 650 nm; in addition, the fluorescence filter 5 blocks (cuts off) the light with a wavelength of 450 - 500 nm, and the cut-off efficiency is better than 0.01%, that is, the light with a wavelength of 450 - 500 nm (the transmitted light wavelength of the filter 3 is within this range) has a transmittance less than 0.01% when passing through the fluorescence filter 5, because the fluorescence wavelength emitted by the sample after being excited by light is 520 - 650 nm, and the fluorescence filter 5 is used to filter out the light not in this range to avoid the interference of the light beam before excitation. The object-side numerical aperture of the imaging lens 6 is greater than 0.1, and the object-image magnification is not less than 0.5X. The image sensor 7 is a area array camera with a resolution of 3 million pixels.Both the light source 1 and the image sensor 7 are connected to the main control unit 8. The main control unit 8 controls the turning on or off of the light source 1 and the image acquisition of the image sensor 7. After the main control unit 8 obtains the image signal of the image sensor 7, it further completes image processing, calculates the characteristics and quantity of cells in the image, and then transmits them to the human-computer interaction module by the main control unit 8 for output through the display screen.

[0115] The counting and typing method in this embodiment is the same as that in Embodiment 2.

[0116] Embodiment 4:

[0117] Figures 12A - 12C Another structure of the detection card of the present invention is shown. The structure of Embodiment 4 is a further improvement on the basis of the structure of Embodiment 1. The difference between its structure and that of Embodiment 1 lies in:

[0118] In this embodiment, there are two detection areas 25, namely the first detection area 51 and the second detection area 52, and the two detection areas are independent of each other (i.e., set at intervals) and connected. The two detection areas are connected by the diversion groove area 26. In this embodiment, both detection areas are rectangles combined with rounded corners and right angles, and the thicknesses are H 检1 , H 检2 , and H 检1 , H 检2 are not equal. For example Figure 12C , the thickness H 检1 of the first detection area 51 is small, and the depth of field of the detection surface formed by the liquid sample to be detected in the first detection area 51 is small, and the spreading area of the sample per unit volume is large, which can be used for precise discrimination of cell types; the thickness H 检1 of the second detection area 52 is large, and the depth of field of the detection surface formed by the liquid sample to be detected in the second detection area 52 is large, and the sample carrying capacity per unit area is large, which is suitable for the overall precise measurement of the number of cells. This embodiment sets two detection areas 51 and 52 with different thicknesses to simultaneously take into account the precision measurement of cell counting and typing. The two detection areas 51 and 52 are connected by the diversion groove area 26 with uniform thickness. Of course, the thicknesses H 检1 , H 检2 of the two detection areas are both smaller than the thickness H 导 of the diversion groove area.

[0119] Of course, multiple detection areas 51, 52 can also have the same thickness, and this thickness value is smaller than the thickness value of the diversion groove area 26. By setting different detection areas, it is helpful to analyze the accuracy and consistency of the detection results of each detection area.

[0120] Other structures of Embodiment 4 are the same as those of Embodiment 1 and will not be elaborated here.

[0121] Embodiment 5:

[0122] This embodiment uses the detection card of Embodiment 1 or Embodiment 4. The human-machine interaction module and the main control unit are the same as those in Embodiment 2. The counting and typing of white blood cells in this embodiment are jointly completed by fluorescence images and scattered light images. The photoelectric detection module is as Figure 3 shown, and includes a fluorescence imaging block and a scattered light imaging block. Among them,

[0123] The fluorescence imaging block sequentially includes a light source 1, a collimating lens 2, a filter 3, a fluorescence filter 5, an imaging lens 6, and an image sensor 7 according to the optical path direction, that is, the light source 1, the collimating lens 2, the filter 3, the fluorescence filter 5, the imaging lens 6, and the image sensor 7 are sequentially arranged on a straight line; a detection card 4 is provided between the filter 3 and the fluorescence filter 5; the main control unit is electrically connected to the light source 1 and the image sensor 7.

[0124] The light source 1 is a blue LED with a wavelength of 470 - 490 nm. The light source 1 is placed at the rear focal point of the collimating lens 2 (with the direction of light propagation as the front), and the light emitted by the light source 1 forms parallel light after passing through the collimating lens 2. The parallel light is filtered by the filter 3 and then irradiated on the detection card 4. The filter 3 is a band-pass filter with a transmission wavelength of 470 - 490 nm. The target cells in the detection card 4 emit fluorescence under the excitation of the light beam irradiation. The fluorescence is sequentially focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6. The target wavelength of the fluorescence filter 5 is 520 - 650 nm, that is, it can transmit light with a wavelength greater than 510 nm; in addition, the fluorescence filter 5 blocks light with a wavelength of 450 - 500 nm (the cut-off efficiency is better than 0.01%), that is, the light with a wavelength of 450 - 500 nm (the transmission wavelength of the light passing through the filter 3 is within this range) has a transmittance less than 0.01% when passing through the fluorescence filter 5, because the fluorescence and scattered light emitted by the sample after being excited by light have a wavelength greater than 510 nm, and the fluorescence filter 5 is used to filter out the light outside this range to avoid the interference of the light beam before excitation. The object-side numerical aperture of the imaging lens 6 is greater than 0.1, and the object-image magnification is not less than 0.5X. The image sensor 7 is a area array camera with a resolution of 3 million pixels. Both the light source 1 and the image sensor 7 are connected to the main control unit 8. The main control unit 8 controls the on or off of the light source 1 and the image acquisition of the image sensor 7; after the main control unit 8 obtains the image signal of the image sensor 7, it further completes image processing, calculates the characteristics and quantity of the cells in the image, and then transmits them to the human-machine interaction module by the main control unit 8 for output through the display screen.

[0125] The scattered light imaging block includes a scattered light illumination source 1', a scattered light lens 2' and a scattered light filter 3' which are arranged in sequence. The scattered light illumination source 1' is a red light LED, and the wavelength of the emitted light is 610nm, which is located at the rear focus of the scattered light lens 2' (with the direction of light propagation as the front). A scattered light filter 3' is arranged in front of the scattered light lens 2', which is used to filter out light with a wavelength less than 600nm. The light emitted by the scattered light illumination source 1' is converged by the scattered light lens 2' to form parallel light, and the parallel light is filtered out by the scattered light filter 3' and then irradiated on the detection card 4. Lymphocytes and monocytes of different sizes will produce scattered light of different light intensities under the irradiation of scattered light. The scattered light passing through the scattered light filter 3' is irradiated on the detection card 4 at an incident angle greater than 45 degrees, so that only the scattered light irradiated on the detection card 4 can enter the imaging lens 6, while the direct transmitted light irradiated on the detection card 4 cannot enter the imaging lens 6. The scattered light reflected by the detection card 4 is sequentially focused and imaged in the image sensor 7 through the fluorescent filter 5 and the imaging lens 6 .

[0126] The scattered light illumination source 1' is also connected to the main control unit 8, and the main control unit 8 controls the on or off of the scattered light illumination source 1'; the light source 1 and the light source 1' are lit in time-sharing manner and cannot be lit at the same time; after the main control unit 8 obtains the image signal of the image sensor 7, it first processes the fluorescence signal, calculates the number of granulocytes and the total number of lymphocytes + monocytes, and then processes the scattered light signal to calculate the number of lymphocytes and monocytes respectively, and then transmits it to the human-computer interaction module through the display screen for output. Figure 6 , Figure 7 They are respectively the fluorescence image and the scattered light image collected by the image sensor 7. The fluorescence image and the scattered light image are used comprehensively to type and count lymphocytes, monocytes and granulocytes.

[0127] The counting typing method of this embodiment specifically comprises the following steps:

[0128] (1) Sampling

[0129] Use whole blood as the test sample, mix the whole blood with the diluent at a ratio of 1:3 to prepare the sample solution to be tested;

[0130] (2) Dyeing

[0131] Add the sample liquid to be tested into the sampling detection cavity of the detection card 4 and let it stand for 2-3 minutes. The sample liquid to be tested will dissolve the reagent in the detection card and react. Under the action of the reagent, the red blood cells in the whole blood will be hemolyzed and the white blood cells will be fluorescently stained.

[0132] (3) Fluorescence excitation and detection

[0133] Insert the detected test card 4 after the reaction into the above-mentioned white blood cell counting and typing instrument, and place the detection area of the test card 4 in the imaging area of the photoelectric detection module; the user starts the detection process through key operations. When using the test card of Example 1, the main control unit 8 drives the light source 1 to light up, and then controls the image sensor 7 to collect fluorescence images, as Figure 8 shown; then, the main control unit 8 turns off the light source 1, drives the scattered light illumination light source 1' to light up, and controls the image sensor 7 to collect scattered light images, as Figure 9 shown. When using the test card of Example 4, one detection area 51 of the test card can be located in the irradiation area (or imaging area) of the light source 1 (fluorescence light source), and the other detection area 52 can be located in the irradiation area (or imaging area) of the scattered light illumination light source 1'. The main control unit 8 can simultaneously drive the light source 1 and the scattered light illumination light source 1' to light up, and control the image sensor 7 to collect fluorescence images and scattered light images.

[0134] (4), Image processing and calculation

[0135] After the image acquisition is completed, the main control unit performs image processing. First, count all the fluorescence points, and use the number of independent fluorescence points as the total number of white blood cells N measured directly; then, for all fluorescence points, count their green light intensity and red light intensity respectively, and calculate a two-dimensional scatter plot according to the green light intensity and red light intensity. Among them, those with a high proportion of red light intensity are granulocytes, counted as N1, and the other part is lymphocytes and monocytes N2, as Figure 7 shown, N = N1 + N2. For those determined to be lymphocytes and monocytes, combine the scattered images to count their scattered light intensity, and the histogram distribution is as Figure 10 shown. The lymphocytes are on the left side (weak scattered light) of the dividing line in the figure, with a quantity of N21, and the monocytes are on the right side (strong scattered light), with a quantity of N22, N2 = N21 + N22.

[0136] Experiment:

[0137] In this experiment, the white blood cell counting and typing instrument of the present invention was compared with the current hospital clinical inspection equipment: 36 outpatient blood samples were selected, and the white blood cell count was 0.3 - 77.5×10 9 cell / L. The hospital clinical inspection equipment is the Sysmex XE-5000 hematology analyzer. The detection method of the white blood cell counting and typing instrument of the present invention is the same as that in Example 2, and the experimental results are shown in Table 1.

[0138] Table 1 Accuracy evaluation results of the white blood cell counting and typing instrument of the present invention

[0139]

[0140] From the results in Table 1, it can be seen that the white blood cell counting and typing instrument of the present invention is at 0.3×10 9cell / L - 77.5×10 9 It has an accurate quantitative detection ability between cell / L and has a consistency of 0.9903 with large clinical fully automatic hematology analyzers.

[0141] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the content of the present invention.

Claims

1. A white blood cell counting and typing instrument, characterized in that, Comprising: A human-computer interaction module for inputting user instructions and outputting detection results; An optoelectronic detection module for detecting and receiving optical signals emitted by a blood sample stained with a nucleic acid stain; A main control unit for transmitting the user instructions input by the human-computer interaction module to the optoelectronic detection module, controlling the optoelectronic detection module to perform detection, processing and calculating the optical signals received by the optoelectronic detection module to complete the counting and typing of white blood cells, and transmitting the detection results of the optoelectronic detection module to the human-computer interaction module; And A detection card, at least for carrying a blood sample, provided with a hollow chamber, and one or more detection areas are provided in the chamber; The detection card is placed in the optoelectronic detection module, and the detection area is located in the imaging area of the optoelectronic detection module; the main control unit is electrically connected to the human-computer interaction module and the optoelectronic detection module; The optoelectronic detection module includes A light source for emitting fluorescence; A collimating lens for refracting the light emitted by the light source into parallel light; A filter for transmitting light in a fixed wavelength range in the light emitted by the light source; A fluorescence filter for transmitting light in a fixed wavelength range in the fluorescence excited by the detection card; An imaging lens for imaging the light transmitted through the fluorescence filter; And An image sensor for receiving the image formed by the imaging lens; The detection card is placed at a position where the transmitted light of the filter can directly shine, the fluorescence filter is placed at a position where the fluorescence excited by the detection card can be received, and both the light source and the image sensor are electrically connected to the main control unit; In the optoelectronic detection module, the light source, the collimating lens, the filter, the fluorescence filter, the imaging lens and the image sensor are arranged at intervals and in parallel in sequence. The detection card is located between the filter and the fluorescence filter and is parallel to the filter. The light emitted by the light source sequentially passes through the collimating lens, the filter, the detection card, the fluorescence filter, the imaging lens and the image sensor; The optoelectronic detection module further includes a beam splitter for reflecting the transmitted light of the filter to the detection area of the detection card and simultaneously transmitting the excited fluorescence of the detection area to the fluorescence filter; The hollow chamber in the detection card is a sampling and detection chamber, which is a semi-open cavity formed by two parallel chamber side walls with a certain gap, and has a detection area, a sampling port, and a diversion channel area connecting the sampling port and the detection area. The thickness H of the detection area 检 is less than the thickness H of the diversion channel area 导 ; The sampling port is in a concave arc shape, and the angle α between the tangent of the downward sliding arc and the horizontal reference plane of the sampling port determines the flow direction of the liquid sample to be measured into the diversion groove area. The value range of the angle α is 15° to 45°.

2. The white blood cell counting and typing instrument according to claim 1, characterized in that The light source, the collimating lens, the filter and the beam splitter are arranged at intervals in sequence. The collimating lens and the filter are parallel, and the angle between the beam splitter and the filter is 45°; the detection card, the beam splitter, the fluorescence filter, the imaging lens and the image sensor are arranged at intervals in sequence. The detection card, the fluorescence filter, the imaging lens and the image sensor are parallel to each other. The angle between the detection card and the filter is 90°, and the angle between the beam splitter and the detection card is 45°; the light emitted by the light source sequentially passes through the collimating lens, the filter, the beam splitter, the detection card, the beam splitter, the fluorescence filter, the imaging lens and the image sensor.

3. The white blood cell counting and typing instrument according to claim 1, wherein The optoelectronic detection module further includes a scattered light imaging block for distinguishing lymphocytes and monocytes in white blood cells; The scattered light imaging block includes a scattered light illumination light source, a scattered light lens, and a scattered light filter that are arranged in parallel at intervals in sequence. The detection area of the detection card is placed at a position where the transmitted light of the scattered light filter can irradiate. The light emitted by the scattered light illumination light source passes through the scattered light lens and the scattered light filter in sequence and irradiates the detection area of the detection card. The scattered light illumination light source is electrically connected to the main control unit.

4. The white blood cell counting and typing instrument according to any one of claims 1-3, characterized in that, An anticoagulant, a hemolytic agent, and a staining agent are attached to the inner wall of the hollow chamber of the detection card; the anticoagulant includes one or more of ethylenediaminetetraacetate, citrate, oxalate, or heparin; the hemolytic agent includes one or more surfactants such as tritonX-100, quaternary ammonium salt, or saponin, and the staining agent includes acridine orange fluorescent dye.

5. The white blood cell counting and typing instrument according to claim 4, characterized in that The sampling port is located at the upper edge opening of the side walls of the two chambers of the sampling and detection chamber, and a sampling notch is provided at the upper edge of one of the side walls of the chamber at the sampling port.

6. The white blood cell counting and typing instrument according to claim 4, characterized in that There are two or more detection areas, and the thicknesses of each detection area are not equal, and they are spatially independent but connected. The detection area with a larger thickness is used for cell counting, and the detection area with a smaller thickness is used for cell typing.

7. A method for white blood cell counting and typing, characterized in that, When using the white blood cell counting and typing instrument according to any one of claims 1-6, it successively includes the steps of diluting whole blood into a sample solution to be measured, adding the sample solution to be measured into the detection card for hemolysis and fluorescence staining, putting the detection card into the white blood cell counting and typing instrument, fluorescence excitation and detection, and image processing and calculation; The fluorescence excitation and detection are specifically three methods: Method 1: The main control unit controls the light source to turn on. The light emitted by the light source passes through a collimating lens, a filter, the detection card, a fluorescence filter, an imaging lens, and an image sensor in sequence. The image sensor transmits the collected fluorescence image to the main control unit; Method 2: The main control unit controls the light source to turn on. The light emitted by the light source passes through a collimating lens, a filter, a beam splitter, the detection card, a beam splitter, a fluorescence filter, an imaging lens, and an image sensor in sequence. The image sensor transmits the collected fluorescence image to the main control unit; Method 3: The main control unit first controls the light source to turn on and turns off the scattered light illumination light source. The light emitted by the light source passes through a collimating lens, a filter, the detection card, a fluorescence filter, an imaging lens, and an image sensor in sequence. The image sensor transmits the collected fluorescence image to the main control unit; then the main control unit controls the scattered light illumination light source to turn on and turns off the light source. The light emitted by the scattered light illumination light source passes through the scattered light lens, the scattered light filter, the detection card, a fluorescence filter, an imaging lens, and an image sensor in sequence. The image sensor transmits the collected scattered light image to the main control unit.

8. The white blood cell counting and typing method according to claim 7, characterized in that, The image processing and calculation are specifically as follows: The main control unit first counts all the fluorescence points, and takes the number of independent fluorescence points as the total number of white blood cells; then it separately counts the green light intensity and the red light intensity of all the fluorescence points. The white blood cells with a higher proportion of red light intensity are taken as the number of granulocytes, and the white blood cells with a higher proportion of green light intensity are taken as the number of lymphocytes and monocytes; Finally, the scattered light signal is processed. The white blood cells with a relatively weak scattered light signal are taken as the number of lymphocytes, and the white blood cells with a relatively strong scattered light signal are taken as the number of monocytes.

9. The white blood cell count typing method according to claim 7 or 8, characterized in that, The specific operation of adding the sample liquid to be tested into the test card is as follows: Immerse the sampling port into the sample liquid to be tested for sampling, and make the sample liquid to be tested flow into the detection area from the diversion groove area and fill it under the action of capillary force. Among them, the capillary force satisfies the following relationship with the thickness of the detection area and the thickness of the diversion groove area: Or Inject the sample liquid to be tested into the sampling and detection cavity through the sampling notch for sampling, and make the sample liquid to be tested flow into the detection area from the diversion groove area and fill it.

Images