A white blood cell counter and a white blood cell counting method

Abstract

The present invention discloses a white blood cell counter and a white blood cell counting method, which include 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 counter of the present invention has the following characteristics: (1) small volume, light weight, convenient to carry and place; (2) using the fluorescence excitation method as the working principle, avoiding the interference of impurities, accurate counting and high precision; (3) simple operation during detection and high detection efficiency; it can make up for the lack of white blood cell detection instruments in current grass-roots medical care, mobile medical care, and emergency rescue medical diagnosis in special environments.

Description

Technical Field

[0001] The present invention relates to the technical field of blood analysis and detection, and particularly to a white blood cell counter and a white blood cell counting 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 play many important roles in the human body. They can phagocytize foreign substances and produce antibodies, have a healing function for body injuries, can resist the invasion of pathogens, and have an immune resistance to diseases, etc. Under normal circumstances, the total number of white blood cells in the human body is relatively stable, and inflammation or other diseases will cause changes in its value, which often show significant changes in the number of white blood cells. 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. Therefore, the change in its value can be used as an indicator of human inflammation or certain diseases. By judging whether the number of white blood cells (WBC) in the blood routine changes, it can be determined whether the body has inflammation or certain diseases. Therefore, counting white blood cells provides an important indicator for modern clinical detection and diagnosis.

[0003] The number of white blood cells, as an indication of certain diseases, has important clinical significance for disease diagnosis. Currently, the main methods for white blood cell counting mainly include the manual microscopy method, the wet chemical detection method, and the blood cell counting method based on dry chemical technology.

[0004] The manual microscopy method is to first hemolyze, that is, to destroy the red blood cells in the blood, then drop it into the counting plate, 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 detected manually and requires professional personnel to complete. Moreover, it takes a long time and is not suitable for the screening of a large number of healthy people. In addition, the analysis results may also vary due to changes in operators, with a large error and not suitable for promotion at the grass-roots level.

[0005] Currently, the wet chemical detection method is mainly used for white blood cell counting detection, which is divided into the Coulter impedance method and the flow cytometry method. The counting principle of the Coulter method is based on the non-conductive properties of blood cells, taking the resistance change caused by the blood cells suspended in the electrolyte solution when passing through the counting aperture as the detection parameter to count the blood cells. Since the resistance change caused is related to the cell size, and different types of blood cells have different volume sizes, the white blood cell counting is realized accordingly. The detection equipment using this method has the characteristics of fast detection speed and high efficiency, but its price is high, the volume is large, and the maintenance requirements are high. It is suitable for clinical laboratory tests in large hospitals and is difficult to be applied to bedside detection, grass-roots diagnosis and treatment, and the treatment of the wounded and sick in special environments.

[0006] The blood cell counting methods based on dry chemical technology mainly include the gradient density centrifugation detection method and the white blood cell staining imaging counting method.

[0007] The gradient density centrifugation method is to perfuse whole blood into a capillary tube. After high-speed centrifugation, different blood cell components are stratified according to density. By imaging and detecting the thickness of different blood cell layers, the detection of various blood cells and the counting of white blood cells are realized. However, the operation steps of this method are relatively cumbersome. Since an additional centrifuge needs to be configured to centrifuge the sample at high speed, it increases the operation difficulty and reduces its portability at the same time.

[0008] The white blood cell staining imaging counting method is represented by the HemoCue white blood cell counter. Its specific detection principle is as follows: First, use a hemolytic agent to dissolve the red blood cells in the blood, and then use a cell staining agent (such as methylene blue, methyl green or gentian violet, etc.) to stain the white blood cells to make the white blood cells colored. Put the stained sample 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 transmitted light. By calculating the number of dark spots, the total number of white blood cells can be calculated. However, the hemolytic counting method adopted by HemoCue has the following disadvantages: First, this method uses the transmission absorption method and is susceptible to interference 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. The above disadvantages result in a low detection accuracy of this method.

[0009] The above methods are all common methods for counting the total number of white blood cells. However, in terms of the professional requirements for operators, equipment volume, price, maintenance, etc. during use, they are not suitable for the needs of primary medical and health institutions and mobile medical care, and there are great limitations in the application process. Summary of the Invention

[0010] The object of the present invention is to address the technical deficiencies existing in the prior art. On the one hand, it provides a white blood cell counter that is easy to carry and used for accurately detecting the number of white blood cells in blood, including

[0011] A human-computer interaction module, which is used to input user instructions and output detection results;

[0012] An optoelectronic detection module, which is used to detect and receive the optical signals emitted by the blood sample stained with a nucleic acid staining agent;

[0013] A main control unit, which is used to transmit the user instructions input by the human-computer interaction module to the optoelectronic detection module, control the optoelectronic detection module to perform detection, process and calculate the optical signals received by the optoelectronic detection module to complete the counting of white blood cells, and transmit the detection results of the optoelectronic detection module to the human-computer interaction module; and

[0014] A detection card, which is used to carry the blood sample;

[0015] The detection card is placed in the optoelectronic detection module, and the main control unit is electrically connected to the human-computer interaction module and the optoelectronic detection module.

[0016] The photoelectric detection module includes

[0017] a light source for emitting fluorescence;

[0018] a filter for transmitting light within a fixed wavelength range in the light emitted by the light source;

[0019] a beam splitter for reflecting the excited fluorescence of the test card onto the imaging lens;

[0020] an imaging lens for imaging the fluorescence excited by the test card;

[0021] a fluorescence filter for transmitting light within a fixed wavelength range in the imaged fluorescence; and

[0022] an image sensor for receiving the optical signal transmitted through the fluorescence filter;

[0023] The beam splitter is placed at a position where the fluorescence excited by the test card can be irradiated, the imaging lens is placed at a position where the fluorescence reflected by the beam splitter can be received, and both the light source and the image sensor are electrically connected to the main control unit.

[0024] In the photoelectric detection module, the light source, the filter and the beam splitter are arranged at intervals in sequence. The test card is arranged between the filter and the beam splitter. The test card and the filter are parallel, and the included angle between the beam splitter and the filter is 45°; the beam splitter, the imaging lens, the fluorescence filter and the image sensor are arranged at intervals in sequence. The imaging lens, the fluorescence filter and the image sensor are parallel to each other. The included angle between the test card and the fluorescence filter is 90°, and the included angle between the beam splitter and the fluorescence filter is 45°; the light emitted by the light source passes through the filter, the test card, the beam splitter, the imaging lens, the fluorescence filter and the image sensor in sequence.

[0025] The test card has a hollow chamber, and an anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the chamber; preferably, the anticoagulant is one or more of ethylenediaminetetraacetate, citrate, oxalate, heparin, etc.; the hemolytic agent is selected from quaternary ammonium salts, saponin, gentian violet or TritonX-100, etc.; the staining agent is selected from SYTO-9, propidium iodide, acridine orange or Cy5, etc.

[0026] The test card includes a support substrate and a hollow chamber provided on the support substrate. The 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 a detection area, a sampling port and a diversion groove area connecting the sampling port and the detection area. The detection area has a single thickness H 检 and the thickness of the detection area is less than the thickness H of the diversion groove area 导 .

[0027] The sampling port is located at the upper edge opening of the side walls of the two chambers of the sampling and detection cavity, and a sampling notch is provided at the upper edge of one of the side walls of the chamber where the sampling port is located.

[0028] The sampling port is arc-shaped, 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 measured into the diversion channel area. The value range of the included angle α is 15° to 45°.

[0029] The test card further includes a sample processing cavity provided on the support substrate, which serves as a storage container for dry or liquid reagents and a mixing operation container for the reagents and blood samples, so that the test card can also be used for pre-treatments such as mixing and staining of blood samples.

[0030] In a second aspect, the present invention provides a white blood cell counting method for quickly detecting the number of white blood cells. Using the above-mentioned white blood cell counter, it sequentially includes steps such as mixing the whole blood of the blood sample into a sample liquid to be measured, adding the sample liquid to be measured into the test card for hemolysis and fluorescence staining, putting the test card into the white blood cell counter, fluorescence excitation and detection, image processing and calculation, etc.

[0031] Preferably, the fluorescence excitation and detection are specifically as follows:

[0032] The main control unit controls the light source to turn on, and the light emitted by the light source sequentially passes through the filter, the test card, the spectroscope, the imaging lens, the fluorescence filter, and the image sensor. The image sensor transmits the collected fluorescence image to the main control unit; or

[0033] Preferably, 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.

[0034] The step of adding the sample liquid to be measured into the test card is specifically as follows: Immerse the sampling port into the sample liquid to be measured for sampling, so that the sample liquid to be measured flows into the detection area from the diversion channel area under the action of capillary force and fills it. Among them, the capillary force satisfies the following relationship with the thickness of the detection area and the thickness of the diversion channel area:

[0035]

[0036] Or,

[0037] Inject the sample liquid to be measured into the sampling and detection cavity through the sampling notch for sampling, so that the sample liquid to be measured flows into the detection area from the diversion channel area and fills it.

[0038] The white blood cell counter of the present invention has the following three characteristics: (1) small in size, light in weight, convenient to carry and place; (2) using the fluorescence excitation method as the working principle, avoiding the interference of impurities, accurate in counting and high in precision; (3) simple in operation during detection and high in detection efficiency; it can make up for the lack of white blood cell detection instruments in primary medical care, mobile medical care and emergency rescue medical diagnosis in special environments at present.

[0039] The detection card used in the white blood cell counter of the present invention can complete the processing and detection of samples. There are reaction grooves and detection areas on the detection card. When in use, reagents and samples can be added to the reaction grooves to complete the processing of samples, and then the processed samples are dropped into the sample adding port to complete the processing of samples, without the need for additional sample processing containers. Brief Description of the Drawings

[0040] Figure 1 The following shows the schematic diagram of the external structure of the white blood cell counter of the present invention;

[0041] Figure 2 The following shows the schematic diagram of the internal structure of the white blood cell counter of the present invention;

[0042] Figure 3 The following shows the schematic diagram of the principle of the white blood cell counter of the present invention;

[0043] Figure 4 The following shows the flowchart of the white blood cell counting method of the present invention;

[0044] Figure 5 The following shows the photo of white blood cells after fluorescence staining by the white blood cell counting method of the present invention;

[0045] Figure 6 The following shows the line graph of the accuracy analysis of the white blood cell counting method of the present invention;

[0046] Figures 7A - 7C The following shows the schematic diagram of the structure of the detection card in the present invention. Detailed Embodiments

[0047] 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 count white blood cells, that is, staining the cells in blood with a fluorescent dye that stains 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 each fluorescent dot corresponding to one cell.

[0048] On this basis, the present invention provides a white blood cell counter, such as Figure 1 and Figure 2As shown, it mainly includes a human-computer interaction module II, a main control unit 8, a photoelectric detection module I, and a detection card 4.

[0049] The detection card 4 is a carrier for white blood cell counting, used to carry blood samples, and can also be used for pretreatment of blood samples such as mixing and staining. The detection card has a hollow chamber, and anticoagulant, hemolytic agent, and staining agent are attached to the inner wall of the chamber; the anticoagulant is one or more of ethylenediaminetetraacetate, citrate, oxalate, heparin, etc.; the hemolytic agent is selected from quaternary ammonium salts, saponin, gentian violet, TritonX-100, etc.; the staining agent is selected from SYTO-9, propidium iodide, acridine orange, Cy5, etc. The detection card 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 a detection area, a sampling port, and a diversion groove area connecting the sampling port and the detection area. The sampling port is arc-shaped, and the 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 measured into the diversion groove area, and the value range of the angle α is 15° to 45°. The detection card 4 also includes a sample processing chamber provided on the support substrate, which serves as a storage container for dry or liquid reagents and a mixing operation container for reagents and liquid samples to be measured.

[0050] The human-computer interaction module II mainly includes a display screen 11 and function keys 12. The instrument also includes a housing 14 and a detection card door 13. Among them, the housing 14 is used to protect the internal components of the instrument, the detection card door 13 is used to put the detection card 4, and the display screen 11 and function keys 12 complete the input of user instructions and the output of detection results; the function keys 12 include a power key, a detection key, and a return key. The power key is used to control the startup and shutdown of the instrument, the detection key is used to start the detection program, and the return key is used to return to the main page. The components of the main control unit 8 and the photoelectric detection module I are assembled in the housing. The display screen 11 and function keys 12 are installed on the surface of the housing and are electrically connected to the main control unit. The detection card door 13 is a drawer-type structure, provided with a sample tray, assembled on one side of the housing and can extend into the housing 14. When the instrument is in use, the detection card 4 is placed on the sample tray and pushed into the housing 14 so that the detection area of the detection card 4 is facing the light source of the photoelectric detection module and is located in its imaging area.

[0051] 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 Youshan Electronic Technology Co., Ltd., electrically connected to the function keys and display screen on the human-computer interaction module, and electrically connected to the photoelectric detection module. After the human-computer interaction module sends 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 sample images, the image processing of the acquired sample images, and the conversion of the digital signal of sample counting into a readable result and feedback it to the display screen of the human-computer interaction module.

[0052] The photoelectric detection module includes a light source 1, a filter 2, a beam splitter 3, an imaging lens 5, a fluorescence filter 6, and an image sensor 7. The main control unit 8 is electrically connected to the light source 1 and the image sensor 7. Among them,

[0053] The light source 1 is an LED light source, which is located directly below the detection area of the test card 4, and the vertical distance from the test card 4 is 10 - 30 mm; a filter 2 is provided between the light source 1 and the test card 4. The light emitted by the light source 1 irradiates the detection area of the test card 4 after passing through the filter 2. The center wavelength of the transmission spectrum of the filter 2 is the same as the center wavelength of the light emitted by the light source 1, and it can filter out stray light other than the light emitted by the light source 1.

[0054] As Figure 3 shown, a beam splitter 3 is provided directly above the test card 4, and the angle between the beam splitter 3 and the light emitted by the light source 1 is 45°. The white blood cells in the blood sample in the detection area of the test card 4 emit fluorescence under the irradiation and excitation of the light source 1, and the fluorescence irradiates onto the beam splitter 3. The beam splitter 3 reflects the fluorescence emitted by the white blood cells and transmits the light of the light source, so that the fluorescence is reflected into the imaging lens, and the light emitted by the light source will not enter the imaging lens. Starting from the beam splitter 3, an imaging lens 5, a fluorescence filter 6, and an image sensor 7 are arranged in sequence in the direction 90° to the light emitted by the light source 1. The fluorescence excited by the test card 4 is polarized by 90° by the beam splitter 3, and then the optical signal is focused into the image sensor 7 through the imaging lens 5 and the filter 6. The image sensor 7 is a area array image sensor with a resolution greater than 3 million pixels.

[0055] 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 common quantity of lymphocytes and monocytes, and then transmits them to the human-computer interaction module by the main control unit 8 for output through the display screen.

[0056] The usage method of the white blood cell counter of the present invention is as follows: When using this white blood cell counter, the user clicks the detection function button on the touch screen, and presses the right test card compartment door inward according to the instrument prompt, and the compartment door will automatically unlock and pop out the sample tray; place the test card on the sample tray, and then push the test card compartment door to close, that is, complete the addition of the sample. At this time, click the confirmation button on the touch screen to start the detection process.

[0057] The main control unit drives to light up the light source, and then controls the image sensor to collect the fluorescence image of the sample; after the fluorescence image is collected, the fluorescence light source is turned off, the scattered light illumination light source is lit, and the image sensor is synchronously controlled to collect the scattered image of the sample, and then the scattered light illumination light source is turned off. After the fluorescence image and the scattered image are collected, the main control unit performs image processing and outputs the detection result on the touch screen display.

[0058] The present invention also provides a white blood cell counting method, as Figure 4 shown, including the following steps:

[0059] (1), Sampling and pretreatment

[0060] Preparation of the sample: Add 10 μl - 20 μl of freshly collected whole blood sample into the sample processing chamber 23 of the test card, and mix until the blood sample becomes semi-transparent to obtain the sample liquid to be tested;

[0061] (2), Staining

[0062] Add the sample liquid to be tested into the detection area of the test card 4 and let it stand for 2 - 3 minutes. The sample liquid to be tested dissolves the reagent in the detection area of the test card and reacts, and under the action of the reagent, hemolysis of red blood cells and fluorescence staining of white blood cells in the whole blood are completed; the reagent in the detection area of the test card includes anticoagulants, hemolyzing agents, and staining agents. The anticoagulant is one or more of ethylenediaminetetraacetate salts, citrates, oxalates, heparin, etc.; the hemolyzing 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;

[0063] (3), Fluorescence excitation and detection

[0064] Insert the reacted test card 4 into the above-mentioned white blood cell counter, 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, and the main control unit 8 drives to light up the illumination light source, and then controls the image sensor 7 to collect the fluorescence image.

[0065] (4), Image processing and calculation

[0066] 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 total number of white blood cells directly measured.

[0067] The following further illustrates the content of the present invention in more detail with specific embodiments, and further elaborates on the present invention, but these embodiments are by no means a limitation to the present invention.

[0068] Embodiment 1:

[0069] Figures 7A - 7C This is a structural example of the test card of the present invention, which is in the form of a slit microfluidic chip structure. Figures 7A - 7CIn 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 handheld part of the slit microfluidic chip, and its design is in a shape suitable for being held. For example, in the first embodiment, the handheld part is rectangular, and the sampling and detection cavity 22 with an arc-shaped edge extends at 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 groove area 26 connecting the sampling port 27 and the detection area 25. Among them:

[0070] 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 way of active suction through the sampling port 27.

[0071] 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; the detection area 25 has a single thickness H 检 , such as Figure 7B shown, when the liquid sample enters the detection area 25, a detection surface can be formed. The thickness range of the detection area 25 is generally 60 - 120 μm.

[0072] The diversion groove 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 groove area 26 is generally 120 μm to 500 μm. As Figure 7C shown, the thickness of the detection area 25 is less than the thickness of the diversion groove area 26, and the liquid sample enters from the sampling port 27 and is uniformly and quickly introduced into and fills the entire sampling and detection cavity 22 through the flow path formed by the diversion groove area 26.

[0073] The thickness of the detection area 25 and the thickness of the diversion groove area 26 determine the flow state of the liquid sample to be tested in the diversion groove area 26 and the spreading state in the detection area 25. The liquid sample to be tested enters the diversion groove 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:

[0074]

[0075] In order to ensure that the liquid sample continuously flows from the diversion groove area 26 into the detection area 5 under the action of capillary force and fills the sampling and detection cavity 22, it is required that the capillary force is greater than zero. The capillary force and the thickness H 检 of the detection area, the thickness H 导 of the diversion groove area have the following relationship:

[0076]

[0077] There is one detection area 25, which is located in the sampling and detection cavity 22 and has a single thickness H 检 , and its shape is a rounded rectangle.

[0078] Furthermore, at the upper end edge of one of the side walls 24 of the two chambers, a sampling notch 29 is provided at the sampling port 27, which is convenient for adding samples through injection at the sampling notch 29. The sampling port 27 and the sampling notch 29 are both compatible with the liquid sampling modes of active injection and passive suction. The sampling port 27 is in a concave arc shape, and the angle α between the tangent of its downward sliding arc (the left arc in the figure) and the horizontal reference plane of the sampling port 27 (see Figure 7A ), this angle determines the flow direction of the liquid sample to be measured into the diversion groove area 26, and the preferred range of the angle α is 15° to 45°, so as to ensure that the liquid sample to be measured spontaneously flows into and fills the sampling and detection cavity 22 in a predetermined manner.

[0079] The sampling port 27 is in an arc shape, and the angle α between the tangent of its downward sliding arc and the horizontal reference plane of the sampling port 27 (see Figure 7A ) determines the flow direction of the liquid sample to be measured into the diversion groove area 26, and the preferred range of the angle α is 15° to 45°, so as to ensure that the liquid sample to be measured spontaneously flows into and fills the sampling and detection cavity 22 in a predetermined manner.

[0080] The structural design of this first embodiment is suitable for cell counting detection with a small number of cells per unit volume or a low type abundance, or application scenarios that only require cell counting. According to the application scenarios, a detection area 25 with a single thickness is selected, and a suitable thickness H is set 检 , in order to perform high-precision measurement of the number of cells or high-precision analysis of individual cell types.

[0081] Of course, in this embodiment, a detection area 25 can 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 that drives the liquid sample to be measured into the sampling and detection cavity 22 and the relationship with the respective thicknesses 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 are formed, and data processing of cells in detection areas with different thicknesses can take into account the precision measurement of cell counting at the same time.

[0082] Embodiment 2:

[0083] The detection card of this embodiment is the same as that of the first embodiment, and the staining agent is SYTO-9.

[0084] The human-computer interaction module II mainly includes a display screen and function buttons, and is responsible for the input of user instructions and the output of detection results; the function buttons include a power switch, a detection button, and a return button. The power switch is used to control the startup and shutdown of the instrument, the detection button is used to start the detection program, and the return button is used to return to the main page.

[0085] The main control unit 8 is responsible for controlling the working process of the instrument, processing and analyzing the collected images. The 4412 type core control board of FriendlyARM is selected, which is electrically connected to the function buttons 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 on or off of the light source in the optoelectronic detection module, the acquisition of sample images, the image processing of the collected sample images, and converts the digital signal of sample counting into a readable result and feeds it back to the display screen of the human-computer interaction module.

[0086] The optoelectronic detection module I, as Figure 3 shown, is composed of a light source 1, a filter 2, a beam splitter 3, an imaging lens 5, a fluorescence filter 6, an image sensor 7, etc. The light source 1, the filter 2, and the beam splitter 3 are arranged in sequence on a vertical straight line along the light propagation direction. The beam splitter 3, the imaging lens 5, the fluorescence filter 6, and the image sensor 7 are arranged in sequence on another horizontal straight line. The vertical straight line where the light source 1 and the filter 2 are located is perpendicular to the horizontal straight line where the imaging lens 5, the fluorescence filter 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 3 is located. The angle between the beam splitter 3 and the horizontal straight line is 45°, and the angle between the beam splitter 3 and the vertical straight line is 45°. The detection card 4 is located on the vertical straight line where the light source 1 and the filter 2 are located, between the beam splitter 3 and the filter 2. The light filtered by the filter 2 irradiates on the detection card 4 and excites fluorescence. The beam splitter 3 can reflect the excited fluorescence onto the imaging lens 5, while the light directly emitted by the light source will not be reflected, thus avoiding the interference of the light emitted by the light source on the fluorescence imaging.

[0087] The light source 1 is an LED light source with a central wavelength of 480nm and a bandwidth of 30nm; the central wavelength of the filter 2 is also 480nm. The object-side numerical aperture of the imaging lens 5 is greater than 0.1, and the object-image magnification is not less than 0.5X. The fluorescence wavelength excited on the detection card 4 is 530nm, the central wavelength of the fluorescence filter 6 is 540nm, the transmittance of light with wavelengths between 520nm - 550nm is greater than 80%, and the transmittance of light with wavelengths less than 500nm is less than 0.01%. Therefore, the fluorescence filter 6 can filter out the light emitted by the light source 1 and avoid the influence of the light emitted by the light source on the sample fluorescence imaging.

[0088] The counting method of this embodiment includes the following steps:

[0089] (1), Sampling and preprocessing

[0090] Preparation of sample: Add 10 μl - 20 μl of freshly collected whole blood sample into the sample processing chamber of the test card, and mix well until the blood sample becomes semi - transparent to obtain the sample solution to be tested;

[0091] (2), Staining

[0092] Aspirate 5 μl - 20 μl of the sample solution to be tested and add it into the detection area of the test card until the detection area is full, then let it stand for 2 - 3 minutes. The sample solution to be tested dissolves the reagent in the detection area of the test card and reacts, and under the action of the reagent, hemolysis of red blood cells and fluorescence staining of white blood cells in the whole blood are completed; The anticoagulant is heparin, the hemolytic agent is glacial acetic acid, and the staining agent is SYTO9 dye;

[0093] (3), Fluorescence excitation and detection

[0094] Click the detection function button on the instrument touch screen, press the right - hand test card compartment door inward with your finger, the compartment door will be automatically unlocked and pop out. Place the test card on the sample holder, then push the test card compartment door to close, insert the test card into the above - mentioned white blood cell counter, and place the detection area of the test card in the imaging area of the photoelectric detection module; The user starts the detection process through key operations. The main control unit drives the illumination light source to light up, and then controls the image sensor to collect fluorescence images, as Figure 5 shown.

[0095] (4), Image processing and calculation

[0096] 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 directly measured total number of white blood cells.

[0097] Experiment 1:

[0098] In this experiment, the white blood cell counter of the present invention is compared with the current hospital clinical inspection equipment: Select 36 outpatient blood samples, and the white blood cell count is 0.3 - 77.5×10 9 cell / L. The hospital clinical inspection equipment is Sysmex XE - 5000 hematology analyzer of Sysmex. The detection method of the white blood cell counter of the present invention is the same as that in Example 2, and the experimental results are shown in Table 1.

[0099] Table 1 Accuracy evaluation results of the white blood cell counter of the present invention

[0100]

[0101] As can be seen from the results in Table 1, the white blood cell counter of the present invention is in the range of 0.3×10 9 cell / L - 77.5×10 9It has an accurate quantitative detection ability between cells / L and has a consistency of 0.9903 with large clinical fully automatic hematology analyzers. See Figure 6 .

[0102] 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 counter, characterized in that, including a human-computer interaction module for inputting user instructions and outputting detection results; a photoelectric 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 photoelectric detection module, controlling the photoelectric detection module to perform detection, processing and calculating the optical signals received by the photoelectric detection module to complete the white blood cell count, and transmitting the detection results of the photoelectric detection module to the human-computer interaction module; and a detection card for carrying the blood sample; 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 detection card has a hollow chamber, and anticoagulant, hemolytic agent and stain are attached to the inner wall of the chamber; the anticoagulant includes one or more of ethylenediaminetetraacetate, citrate, oxalate or heparin; the hemolytic agent includes one or more of quaternary ammonium salt, saponin, gentian violet or TritonX-100; the stain includes one or more of SYTO-9, propidium iodide, acridine orange or Cy5; The detection card includes a support substrate and a hollow chamber provided on the support substrate. The 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 a detection area, a sampling port, and a diversion groove area connecting the sampling port and the detection area. The detection area has a single thickness H 检 , and the thickness of the detection area is less than the thickness H of the diversion groove area 导 ; the sampling port is located at the upper edge opening of the side walls of the two chambers of the sampling and detection cavity, and a sampling notch is provided at the upper edge of one of the side walls of the chamber at the sampling port; the sampling port is arc-shaped, and the 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 measured into the diversion groove area, and the value range of the angle α is 15° to 45°.

2. The white blood cell counter according to claim 1, characterized in that, the photoelectric detection module includes a light source for emitting fluorescence; a filter for transmitting light in a fixed wavelength range in the light emitted by the light source; a beam splitter for reflecting the excitation fluorescence of the detection card onto the imaging lens; an imaging lens for imaging the fluorescence excited by the detection card; a fluorescence filter for transmitting light in a fixed wavelength range in the imaging fluorescence; and an image sensor for receiving the optical signal transmitted by the fluorescence filter; the beam splitter is placed at a position where the fluorescence excited by the detection card can be irradiated, the imaging lens is placed at a position where the fluorescence reflected by the beam splitter can be received, and the light source and the image sensor are both electrically connected to the main control unit.

3. The white blood cell counter according to claim 2, wherein, in the photoelectric detection module, the light source, the filter and the beam splitter are arranged at intervals in sequence, the detection card is arranged between the filter and the beam splitter, the detection card and the filter are parallel, and the angle between the beam splitter and the filter is 45°; the beam splitter, the imaging lens, the fluorescence filter and the image sensor are arranged at intervals in sequence, the imaging lens, the fluorescence filter and the image sensor are parallel to each other, the angle between the detection card and the fluorescence filter is 90°, and the angle between the beam splitter and the fluorescence filter is 45°; the light emitted by the light source passes through the filter, the detection card, the beam splitter, the imaging lens, the fluorescence filter and the image sensor in sequence.

4. The white blood cell counter according to claim 1, wherein, the detection card further includes a sample processing chamber provided on a support substrate, which serves as a storage container for dry or liquid reagents and a mixing operation container for the reagents and the blood sample, so that the detection card can also be used for mixing and / or staining pretreatment of the blood sample.

5. A method for white blood cell counting, characterized in that, Using the white blood cell counter according to any one of claims 1-4, it successively includes the steps of mixing whole blood of a blood sample into a sample liquid to be tested, adding the sample liquid to be tested into a detection card for hemolysis and fluorescence staining, putting the detection card into the white blood cell counter, fluorescence excitation and detection, and image processing and calculation; The fluorescence excitation and detection specifically are: The main control unit controls the light source to turn on, and the light emitted by the light source successively passes through a filter, a detection card, a beam splitter, an imaging lens, a fluorescence filter, and an image sensor, and the image sensor transmits the collected fluorescence image to the main control unit; or The image processing and calculation specifically are: The main control unit first counts all fluorescence dots, and takes the number of independent fluorescence dots as the total number of white blood cells.

6. The white blood cell counting method according to claim 5, characterized in that The step of adding the sample liquid to be tested into the detection card specifically is: dipping the sampling port into the sample liquid to be tested for sampling, so that the sample liquid to be tested flows into the detection area from the diversion groove area under the action of capillary force and fills 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: Or, Inject the sample liquid to be tested into the sampling and detection cavity through the sampling notch for sampling, so that the sample liquid to be tested flows into the detection area from the diversion groove area and fills it.

Images