CN111912770A - Handheld leucocyte counting instrument and leucocyte counting method - Google Patents
Handheld leucocyte counting instrument and leucocyte counting method Download PDFInfo
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Abstract
The invention discloses a handheld leucocyte counting instrument and a leucocyte counting method. The invention relates to a handheld leucocyte counter, which comprises a shell suitable for being held by hand, and a photoelectric detection module, a main control unit, a man-machine interaction module detection card door, a bracket, a detection card and the like which are assembled together, wherein the detection card is in a slit microfluidic chip structure and is used for counting leucocytes by a fluorescence excitation principle. The instrument has small volume, light weight and convenient carrying and installation, the counting method can avoid the interference of impurities, has accurate counting, high precision, simple detection operation and high detection efficiency, and is particularly suitable for detecting the leucocytes in primary medical treatment, mobile medical treatment and emergency rescue medical diagnosis in special environments.
Description
Technical Field
The invention relates to the technical field of blood analysis and detection, in particular to a handheld leucocyte counting instrument and a leucocyte counting method.
Background
Blood cells in the human body are divided into three categories: white blood cells, red blood cells, platelets. Leukocytes play a major role in the human body, phagocytize foreign substances and produce antibodies, have a healing function on body injuries, can resist pathogen invasion, and produce immune resistance to diseases. Under normal conditions, the total number of leukocytes in a human body is relatively constant, and inflammation or other diseases cause changes in their numbers, often manifested by significant changes in the number of leukocytes. That is, the total number of leukocytes is changed due to inflammation or other diseases, so the change of the value can be used as an index of inflammation or some diseases of the human body, and whether the inflammation or some diseases of the body occurs is judged by whether the number of leukocytes (WBC) in the blood routine is changed. Therefore, counting leukocytes is an important index for modern clinical detection and diagnosis.
The number of leukocytes as an indicator of certain diseases is of great clinical significance in disease diagnosis. Current methods for white blood cell counting mainly include manual microscopy, wet chemistry assays, and dry chemistry based blood cell counting methods.
The manual microscopy method comprises hemolysis, i.e. breaking red blood cells in blood, dripping into a counting disc, counting the number of white blood cells in a certain range under a microscope, and converting into the number of white blood cells in each liter of blood. The method is manually used for detection, needs professional personnel for completion, is long in time consumption, is not suitable for screening of a large number of healthy people, and is not suitable for popularization in the basic level, and analysis results are different due to changes of operators and are large in error.
At present, the leukocyte count detection mainly uses a wet chemistry detection method which is divided into a Coulter electrical impedance method and a flow cytometry method. The coulter method counts blood cells based on their nonconductive properties by using the resistance change caused by the blood cells suspended in an electrolyte solution as a detection parameter when passing through a counting well, and counts leukocytes by using the volume of different blood cells different in size due to the resistance change caused by the change in cell size. The detection equipment using the method has the characteristics of high detection speed and high efficiency, but has high price, large volume and high maintenance requirement, is suitable for large hospitals to carry out clinical tests, and is difficult to be suitable for bedside detection, basic diagnosis and treatment and the treatment of patients in special environments.
The dry chemical technology-based blood cell counting method mainly comprises a gradient density centrifugation detection method and a white blood cell staining imaging counting method.
The gradient density centrifugation method is that whole blood is filled into a capillary tube, after high-speed centrifugation, different blood cell components are layered according to density, and the thickness of different blood cell layers is detected through imaging, so that the detection of various blood cells and the counting of leucocytes are realized. The method has more complicated operation steps, and a centrifuge is additionally arranged to centrifuge the sample at a high speed, so that the operation difficulty is additionally increased, and the portability is reduced.
The leucocyte staining imaging counting method is represented by a Hemocue leucocyte counter, and the specific detection principle is as follows: dissolving red blood cells in blood with hemolytic agent, staining white blood cells with cell stain (such as methylene blue, methyl green or gentian violet) to stain white blood cells, coloring white blood cells, placing the stained sample under transmitted light, wherein the color of the stained sample is deepened after the white blood cells are stained, the stained sample can be represented as dark spots on an image under the irradiation of the transmitted light, and the total number of the white blood cells can be calculated by calculating the number of the dark spots. However, the hemolytic counting method employed by HemoCue has the following disadvantages: firstly, the method uses a transmission absorption method, which is easily interfered by impurities; secondly, the detection time window is short, the detection must be strictly controlled within 2-10min, the dissolution of the red blood cells is incomplete when the time is too short, and the white blood cells can be dissolved when the time is too long. The above disadvantages result in a low detection accuracy of the method.
The methods are common methods for counting the total number of the white blood cells, but the requirements on the professional performance of operators in use, equipment volume, price, maintenance and the like are not suitable for the requirements of primary medical treatment, mobile medical treatment and emergency rescue medical diagnosis in special environments, and the methods have great limitations in the application process.
Disclosure of Invention
The present invention is directed to overcoming the technical deficiencies of the prior art in providing, in a first aspect, a portable hand-held white blood cell counter for accurately measuring the number of white blood cells in blood, comprising
The human-computer interaction module is used for inputting a user instruction and outputting a detection result;
the photoelectric detection module is used for detecting and receiving an optical signal emitted by the blood sample dyed by the nucleic acid dyeing agent;
the main control unit is used for transmitting a user instruction input by the human-computer interaction module to the photoelectric detection module, controlling the photoelectric detection module to detect, processing an optical signal received by the photoelectric detection module, calculating the count of white blood cells, and transmitting a detection result of the photoelectric detection module to the human-computer interaction module; and
the detection card is used for bearing the blood sample;
still including being suitable for handheld casing, photoelectric detection module and main control unit all settle inside the casing, and the human-computer interaction module is established at the casing upper surface, and a side of casing is equipped with the detection card hatch door, can take out or put into the casing, and the detection card hatch door is equipped with the bracket that stretches into to the casing inside in the casing, is equipped with on the bracket with detection card size matched with detection card cabin, detects the screens in the photoelectric detection module, and the main control unit is connected with human-computer interaction module and photoelectric detection module electricity.
A lock head is arranged at one end of the bracket extending into the shell, and a self-locking buckle is arranged at the position of the inner wall of the shell corresponding to the lock head and used for locking and popping the bracket; preferably, the inner wall of the bottom surface of the shell is provided with a sliding rail matched with the bracket.
The photodetection module includes:
a light source for emitting fluorescent light;
a filter for transmitting light of a fixed wavelength range among the light emitted from the light source;
the light splitting sheet is used for reflecting the excited fluorescence of the detection card to the imaging lens;
the imaging lens is used for imaging the fluorescence excited by the detection card;
a fluorescence filter for transmitting light of a fixed wavelength range in the imaging fluorescence; and
the image sensor is used for receiving the optical signal transmitted by the fluorescent filter;
the light splitting sheet is arranged at a position where the fluorescence excited by the detection card can irradiate, the imaging lens is arranged at a position where the fluorescence reflected by the light splitting sheet can be received, and the light source and the image sensor are both electrically connected with the main control unit.
The photoelectric detection module is characterized in that a light source, an optical filter and a light splitting sheet are sequentially arranged at intervals, a detection card is arranged between the optical filter and the light splitting sheet, the detection card is parallel to the optical filter, and an included angle between the light splitting sheet and the optical filter is 45 degrees; the light splitting piece, the imaging lens, the fluorescent light filter and the image sensor are sequentially arranged at intervals, the imaging lens, the fluorescent light filter and the image sensor are parallel to each other, the included angle between the detection card and the fluorescent light filter is 90 degrees, and the included angle between the light splitting piece and the fluorescent light filter is 45 degrees; the light emitted by the light source sequentially passes through the optical filter, the detection card, the light splitting sheet, the imaging lens, the fluorescent optical filter and the image sensor.
The detection card is provided with a hollow cavity, and an anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the cavity; preferably, the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is one or more of surfactant selected from quaternary ammonium salt, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, NP-40, etc.; the stain is one or more of fluorescent dyes such as SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and the like.
The detection card comprises a support substrate and a hollow cavity arranged on the support substrate, the cavity is a sampling detection cavity and is a semi-open cavity formed by two parallel cavity side walls with a certain gap, a flow guide groove area with a detection area, a sampling opening and a communication sampling opening and the detection area is provided, and the detection area has a single thickness HDetection ofAnd the thickness of the detection area is less than the thickness H of the flow guide groove areaGuide tube。
The sampling opening is positioned at the upper end edge opening of the two cavity side walls of the sampling detection cavity, the upper end edge of one cavity side wall positioned at the sampling opening is provided with a sampling notch, the sampling opening is arc-shaped, the included angle alpha between the tangent line of the downward sliding arc line and the horizontal reference surface of the sampling opening determines the flow direction position of the liquid sample to be detected entering the flow guide groove area, and the value range of the included angle alpha is 15-45 degrees.
The detection card also comprises a sample processing cavity arranged on the supporting substrate, and the sample processing cavity is used as a storage container of dry or liquid reagents and a mixing operation container of the reagents and the blood sample, so that the detection card can also be used for pretreatment such as uniform mixing, dyeing and the like of the blood sample.
In a second aspect, the invention provides a portable leukocyte counting method for rapidly detecting the number of leukocytes, which comprises the steps of preparing a blood sample into a sample solution to be detected, adding the sample solution to be detected into a detection card for hemolysis and fluorescent staining, placing the detection card into the handheld leukocyte counting instrument, performing fluorescent excitation and detection, processing and calculating images and the like;
preferably, the fluorescence excitation and detection specifically comprises:
the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the optical filter, the detection card, the light splitting sheet, the imaging lens, the fluorescent optical filter and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit; or
Preferably, the image processing and calculating specifically includes: the main control unit counts all the fluorescence points first, and the number of the independent fluorescence points is used as the total number of the white blood cells.
The step of adding the sample liquid to be detected into the detection card specifically comprises the following steps: the sampling port is immersed in the sample liquid to be tested for sampling, so that the sample liquid to be tested flows into the detection area from the flow guide groove area under the action of capillary force and is filled with the sample liquid, wherein the capillary force and the thickness of the detection area and the thickness of the flow guide groove area satisfy the following relations:
alternatively, the first and second electrodes may be,
and injecting the sample liquid to be detected into the sampling detection cavity through the sampling notch for sampling, so that the sample liquid to be detected flows into the detection area from the diversion groove area and is filled.
The handheld leucocyte counter has the following three characteristics: (1) small volume, light weight, convenient carrying and installation; (2) a fluorescence excitation method is adopted as a working principle, so that the interference of impurities is avoided, the counting is accurate, and the precision is high; (3) the operation is simple during detection, and the detection efficiency is high; can make up the deficiency of the handheld leucocyte detecting instrument in the emergency rescue medical diagnosis in the current primary medical treatment, the mobile medical treatment and the special environment.
The detection card used by the handheld leucocyte counting instrument can complete the treatment and detection of the sample, the detection card is provided with the reaction groove and the detection area, when in use, the reagent and the sample can be added into the reaction groove to complete the treatment of the sample, and then the treated sample is dripped into the sample adding port to complete the treatment of the sample without an additional sample treatment container.
Drawings
FIG. 1 is a schematic view of the external structure of the hand-held white blood cell counter of the present invention;
FIGS. 2A-2B are schematic diagrams of the internal structure of the hand-held white blood cell counter of the present invention;
FIG. 3 is a schematic diagram of the hand-held white blood cell counter of the present invention;
FIG. 4 is a flow chart of a method of counting leukocytes according to the invention;
FIG. 5 is a photograph showing white blood cells after fluorescent staining by the white blood cell counting method of the present invention;
FIG. 6 is a line graph showing the accuracy analysis of the method for counting leukocytes according to the invention;
FIGS. 7A-7C are schematic views of the structure of the detection card of the present invention;
FIG. 8 is a block diagram of a hand-held white blood cell counter according to the present invention;
figure 9 shows a top view of the bracket of the present invention.
Detailed Description
Since red blood cells and plasma in blood have no nucleic acid and white blood cells have nuclei, the present invention uses this distinction to count white blood cells, i.e., cells in blood are stained with a fluorescent dye that stains nucleic acid (e.g., acridine orange, SYTO9, etc.), and red blood cells and plasma have no nucleic acid and therefore no fluorescence; platelets do not produce fluorescence; leukocytes have nuclei and emit fluorescence. By using the principle, red blood cells, plasma and white blood cells can be distinguished, and the total number of the white blood cells is calculated according to one cell corresponding to each fluorescent point.
On the basis, the invention provides a handheld leucocyte counting instrument, which has an external structure schematic diagram shown in fig. 1 and an internal structure shown in fig. 2A, and a structural block diagram shown in fig. 8, and mainly comprises a shell 14, a man-machine interaction module, a main control unit 8, a photoelectric detection module I and a detection card 4.
The detection card 4 is a carrier for counting leukocytes, is used for carrying a blood sample, and can also be used for pretreatment such as mixing and dyeing of the blood sample. The detection card 4 is provided with a hollow cavity, and an anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the cavity; the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is one or more of surfactant selected from quaternary ammonium salt, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, NP-40, etc.; the stain is one or more of fluorescent dyes such as SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and the like. The detection card comprises a support substrate and a hollow cavity arranged on the support substrate, the cavity is a sampling detection cavity, is a semi-open cavity formed by two parallel cavity side walls with a certain gap, and is provided with a detection area, a sampling opening and a flow guide groove area communicated with the sampling opening and the detection area. The detection zone has a single thickness HDetection ofAnd the thickness of the detection area is less than the thickness H of the flow guide groove areaGuide tube. The sampling port is arc-shaped, an included angle alpha between a tangent line of a downward sliding arc line of the sampling port and a horizontal reference plane of the sampling port determines the direction of a liquid sample to be detected entering the flow guide groove area, and the value range of the included angle alpha is 15-45 degrees. The test card 4 further comprises a sample processing chamber disposed on the support substrate as a storage container for dry or liquid reagents and a mixing operation container for mixing the reagents with a liquid sample to be tested.
The shell 14 is mainly used for protecting internal components of the instrument, and mainly comprises a USB interface, a power interface, a battery compartment 20, a detection card compartment door 13, a self-locking buckle 15, a bracket 16 and the like. The USB interface and the power interface are provided on the side wall of the housing 14. A battery compartment 20 is provided inside the housing 14 (see fig. 2B). A test card slot door 13 is provided on the other side wall of the housing 14 as a passage for inserting and removing the test card 4. The inspection card slot door 13 is of a drawer type structure, and as shown in fig. 9, the portion inside the casing 14 is a bracket 16, and the bracket 16 is used for placing the inspection card 4. The upper surface of the bracket 16 is provided with a detection card chamber 12 matched with the size of the detection card 4. One end of the bracket 16 is connected with the detection card cabin door 13, and the other end is provided with a lock head 17. And a self-locking buckle 15 is arranged on the inner wall of the shell 14 and at a position corresponding to the lock head 17 and used for locking and ejecting the bracket. The inner wall of the bottom surface of the housing 14 is provided with a slide rail 18, the shape of the rail of the slide rail 18 is matched with the shape of the two side surfaces of the bracket 16, and the bracket 16 can be pulled out of the housing 14 or pushed into the housing 14 through the slide rail 18. The upper surface of the housing 14 is further provided with a human-computer interaction module (the surface on which the human-computer interaction module is arranged is the upper surface, and the surface opposite to the upper surface is the bottom surface).
The man-machine interaction module mainly comprises a display screen 11 and a function key 19, wherein the display screen 11 and the function key 19 finish the input of user instructions and the output of detection results; the function keys 19 include an on-off key for controlling the start and stop of the instrument, a detection key for starting a detection program, and a return key for returning to the main page.
The main control unit 8 is responsible for controlling the work flow of the instrument, a 4412 type core control panel purchased from Guangzhou friendly electronic technology Co., Ltd is electrically connected with a function key and a display screen on the man-machine interaction module and is electrically connected with the photoelectric detection module, after the man-machine interaction module sends an instruction to the main control unit 8, the main control unit 8 controls the on/off of a light source in the photoelectric detection module, the acquisition of sample images, the image processing of the acquired sample images and the conversion of digital signals of sample counting into readable results and feeds back the readable results to the display screen of the man-machine interaction module.
The photoelectric detection module I comprises a light source 1, an optical filter 2, a light splitting sheet 3, an imaging lens 5, a fluorescent optical 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. Wherein the content of the first and second substances,
the light source 1 is an LED light source and is positioned right below a detection area of the detection card 4, and the vertical distance from the detection card 4 is 10-30 mm; an optical filter 2 is arranged between the light source 1 and the detection card 4. The light emitted from the light source 1 passes through the filter 2 and then irradiates the detection area of the detection card 4. The central wavelength of the transmission spectrum of the optical filter 2 is consistent with the central wavelength of the light emitted by the light source 1, and stray light except the light emitted by the light source 1 can be filtered and removed.
As shown in fig. 3, a light splitting sheet 3 is disposed right above the detection card 4, and an angle between the light splitting sheet 3 and the light emitted from the light source 1 is 45 °. The leucocyte in the blood sample detected by the detection area of the detection card 4 emits fluorescence under the irradiation excitation action of the light source 1, and the fluorescence irradiates the light splitting sheet 3. The light splitter 3 reflects fluorescence emitted from white blood cells and transmits light from the light source, so that the fluorescence is reflected to the imaging lens, and the light emitted from the light source does not enter the imaging lens. An imaging lens 5, a fluorescence filter 6 and an image sensor 7 are sequentially arranged in a direction which takes the light splitting sheet 3 as a starting point and forms an angle of 90 degrees with the light emitted by the light source 1. Fluorescence excited by the detection card 4 is polarized by 90 degrees through the beam splitter 3, and then an optical signal is focused into the image sensor 7 through the imaging lens 5 and the optical filter 6, wherein the image sensor 7 is an area array image sensor, and the resolution is more than 300 ten thousand pixels.
The main control unit 8 and the photoelectric detection module I are assembled in the shell 14, the display screen 11 and the function keys 19 are installed on the surface of the shell 14 and electrically connected with the main control unit 8, the detection card door 13 is of a drawer type structure and is provided with a bracket 16 which is assembled on one side surface of the shell 14 and can extend into the shell 14, the detection card 4 is placed on the detection card cabin 12 of the bracket 16 when the instrument is used, and the detection card is pushed into the shell 14 so that the detection area of the detection card 4 is opposite to the light source of the photoelectric detection module and is positioned in the imaging area of the photoelectric detection module. The light source 1 and the image sensor 7 are both connected with the main control unit 8, and 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 acquires the image signal of the image sensor 7, further image processing is completed, the features and the number of cells in the image are calculated, the total number of white blood cells, namely the number of granulocytes and the common number of lymphocytes and monocytes are respectively obtained, and then the total number of white blood cells is transmitted to the human-computer interaction module by the main control unit 8 and is output through the display screen.
The application method of the leucocyte counter comprises the following steps: when the handheld leucocyte counting instrument is used, a user clicks a detection function button on a touch screen, a finger is prompted to press the detection card cabin door on the right side inwards according to the instrument, and the cabin door is automatically unlocked and pops out of a sample bracket; the detection card is placed on the sample bracket, then the cabin door of the detection card is pushed in and closed, namely, the addition of the sample is completed, at the moment, the confirmation key on the touch screen is clicked, and the detection process is started.
The main control unit drives and lights the light source, and then controls the image sensor to collect a fluorescence image of the sample; and after the fluorescent image is collected, the fluorescent light source is closed, the scattered light illuminating light source is lightened, the image sensor is synchronously controlled to collect the scattered image of the sample, and then the scattered light illuminating light source is closed. And after the fluorescent image and the scattering image are collected, the main control unit processes the images and displays and outputs the detection result on the touch screen.
The present invention also provides a method for counting leukocytes, as shown in fig. 4, comprising the steps of:
(1) sampling and pretreatment
Preparation of the sample: adding 10-20 mul of a freshly collected whole blood sample into a sample processing cavity 23 of the detection card, and uniformly mixing until the blood sample is semitransparent to obtain a sample solution to be detected;
(2) and dyeing the mixture
Adding the sample liquid to be detected into the detection area of the detection card 4, standing for 2-3 minutes, dissolving the reagent in the detection area of the detection card by the sample liquid to be detected, reacting, and completing hemolysis of red blood cells in whole blood and leucocyte fluorescent staining under the action of the reagent; the reagent in the detection card detection area comprises an anticoagulant, a hemolytic agent and a coloring agent, wherein the anticoagulant is one or more of ethylenediamine tetraacetate, citrate, oxalate, heparin and the like; the hemolytic agent is one or more of surfactant selected from quaternary ammonium salt, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, NP-40, etc.; the stain is one or more of fluorescent dyes such as SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and the like;
(3) fluorescence excitation and detection
Inserting the reacted detection card 4 into the leucocyte counting instrument, and placing a detection area of the detection card 4 in an imaging area of a photoelectric detection module; the user starts the detection process through key operation, the main control unit 8 drives and lights the illumination light source, and then the image sensor 7 is controlled to collect the fluorescence image.
(4) Image processing and calculation
After the image acquisition is completed, the main control unit 8 performs image processing: all fluorescence spots were first counted, and the number of independent fluorescence spots was taken as the total number of leukocytes directly measured.
The present invention will be described more specifically and further illustrated with reference to specific examples, which are by no means intended to limit the scope of the present invention.
The first embodiment is as follows:
fig. 7A to 7C are structural examples of the detection card of the present invention, which is in the form of a slit microfluidic chip structure. In the first embodiment shown in fig. 7A-7C, the detection card includes a support substrate 21 and a sampling detection cavity 22 disposed on the support substrate 21, wherein the support substrate 21 is a hand-held portion of the microfluidic chamber, which is designed to be hand-held, and in the first embodiment, the hand-held portion is rectangular, and the front end of the hand-held portion extends to form the sampling detection cavity 22 with an arc-shaped edge; the sampling test chamber 22 may be integrally formed with the support substrate 21, or the sampling test chamber 22 may be bonded to the front end of the support substrate 21. The sampling and detecting cavity 22 is a semi-open cavity formed by two parallel cavity side walls 24 with a certain gap, and comprises a sampling port 27, a sampling gap 29, a detecting region 25 and a flow guide groove region 26 communicated with the sampling port 27 and the detecting region 25, wherein:
the sampling port 27 is located at the opening of the edge of the sampling detection cavity 22, and the sample can be actively sucked through the sampling port 27.
The detection area 25 is positioned in the sampling detection cavity 22, the shape of the detection area 25 can be a rectangle, a square, a trapezoid, a circle or a combination of an arc and other shapes, and each shape can be provided with a round angle, a right angle or a combination of the round angle and the right angle; the detection zone 25 has a single thickness HDetection ofAs shown in FIG. 7B, the entrance of the liquid sample into the detection zone 25 forms a detection surface, and the thickness of the detection zone 25 is generally in the range of 60 to 120. mu.m.
The diversion trench area 26 is located in the sampling detection cavity 22 and is communicated with the sampling port 27 and the detection area 25, and the thickness range of the diversion trench area 26 is generally 120-500 μm. As shown in FIG. 7C, the thickness of the detection region 25 is smaller than that of the flow guide groove region 26, and the liquid sample is introduced from the sampling port 27 into the flow path formed by the flow guide groove region 26 uniformly and rapidly and fills the whole sampling detection cavity 22.
The thickness of the detection area 25 and the thickness of the flow guide groove area 26 determine the flowing state of the liquid sample to be tested in the flow guide groove area 26 and the spreading state in the detection area 25. The liquid sample to be measured enters the diversion trench area 26 through the sampling port 27, the sample suction phase belongs to the pure inertia rising phase under the action of capillary force, and the relation between the volume of the sucked liquid sample to be measured and the thickness of the detection area 25 can be obtained according to the formula 1) of the pure inertia rising phase of capillary flow:
in order to ensure that the liquid sample continuously flows from the flow guide groove region 26 into the detection region 5 and fills the sampling detection cavity 22 under the action of capillary force, the capillary force is required to be larger than zero. Capillary force and thickness H of detection zone 25Detection ofThickness H of the flow guide groove areaGuide tubeThe following relationships exist:
the detection area 25 is provided with one and is positioned in the sampling detection cavity 22 and has a single thickness HDetection ofThe shape is a round-corner rectangle.
Furthermore, a sampling notch 29 is provided on the upper end edge of one of the chamber side walls 24 at the sampling port 27, so that the sample can be conveniently loaded from the sampling notch 29 by injection. The sampling port 27 and the sampling gap 29 are compatible with liquid sampling modes of active sample injection and passive sample suction at the same time.
The sampling port 27 is arc-shaped, the included angle alpha (see fig. 7A) between the tangent line of the downward sliding arc line and the horizontal reference plane of the sampling port 27 determines the direction of the liquid sample to be detected entering the flow guide groove area 26, and the preferred range of the angle alpha is 15-45 degrees, so that the liquid sample to be detected can be ensured to spontaneously flow into and fill the sampling detection cavity 22 in a predetermined manner.
In addition, the holding end of the supporting substrate 21 has a certain thickness (the thickness of the holding end is larger than that of the sampling detection cavity 22), and the supporting substrate 21 is provided with an inverted truncated cone-shaped groove as a sample processing cavity 23 which can be used as a storage container for dry or liquid reagents and a mixing operation container for mixing the reagents and a liquid sample to be detected during detection, and is used for pretreatment of the blood sample.
Example two:
the detection card of the embodiment is the same as the first embodiment, and the staining agent is SYTO-9.
The man-machine interaction module mainly comprises a display screen and a function key, and completes the input of user instructions and the output of detection results; the function keys comprise an on-off key, a detection key and a return key, wherein the on-off key is used for controlling the starting and the closing of the instrument, the detection key is used for starting a detection program, and the return key is used for returning to the main page.
The main control unit 8 is responsible for controlling the work flow of the instrument, processing and analyzing the collected images, a 4412 type core control panel with a friendly arm is selected, is electrically connected with a function key and a display screen on the human-computer interaction module and is electrically connected with 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 a light source in the photoelectric detection module, the collection of sample images, the image processing of the collected sample images and the conversion of digital signals of sample counting into readable results and feeds back the readable results to the display screen of the human-computer interaction module.
The photoelectric detection module, as shown in fig. 3, 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, and the like. The light source 1, the optical filter 2 and the light splitting sheet 3 are sequentially arranged on a vertical straight line along the propagation direction of light, the light splitting sheet 3, the imaging lens 5, the fluorescent filter 6 and the image sensor 7 are sequentially arranged on another horizontal straight line, the vertical straight line where the light source 1 and the optical filter 2 are located is perpendicular to the horizontal straight line where the imaging lens 5, the fluorescent filter 6 and the like are located, the focuses (namely, the feet of the two straight lines) are the positions of the light splitting sheet 3, the included angle between the light splitting sheet 3 and the horizontal straight line is 45 degrees, and the included angle between the light splitting sheet 3 and the vertical straight line is 45 degrees. The detection card 4 is located on a vertical straight line where the light source 1 and the optical filter 2 are located and located between the light splitting sheet 3 and the optical filter 2, light filtered by the optical filter 2 irradiates the detection card 4 to excite fluorescence, the light splitting sheet 3 can reflect the excited fluorescence to the imaging lens 5, light directly emitted by the light source cannot be reflected, and therefore interference of the light emitted by the light source on fluorescence imaging is avoided.
The light source 1 is an LED light source, the central wavelength is 480nm, and the bandwidth is 30 nm; the filter 2 also has a central wavelength of 480 nm. The object space numerical space of the imaging lens 5 is larger than 0.1, and the object magnification is not smaller than 0.5X. The wavelength of fluorescence excited on the detection card 4 is 530nm, the central wavelength of the fluorescence filter 6 is 540nm, the transmittance of light with the wavelength of 520nm to 550nm is greater than 80%, and the transmittance of light with the wavelength less than 500nm is less than 0.01%, so that the fluorescence filter 6 can filter the light emitted by the light source 1, and the influence of the light on the fluorescence imaging of the sample is avoided.
The counting method of the embodiment comprises the following steps:
(1) sampling and pretreatment
Preparation of the sample: adding 10-20 mul of a freshly collected whole blood sample into a sample processing cavity of a detection card, and uniformly mixing until the blood sample is semitransparent, so as to obtain a sample solution to be detected;
(2) and dyeing the mixture
Sucking 5-20 mul of sample liquid to be detected, adding the sample liquid to be detected into the detection area of the detection card until the detection area is full, standing for 2-3 minutes, dissolving the reagent in the detection area of the detection card by the sample liquid to be detected, reacting, and completing hemolysis of red blood cells in whole blood and leucocyte fluorescent staining under the action of the reagent; the anticoagulant is ethylenediamine tetraacetate, the hemolytic agent is selected from Tween80, and the staining agent is SYTO9 dye;
(3) fluorescence excitation and detection
Clicking a detection function button on a touch screen of the instrument, pressing a detection card cabin door on the right side inwards by a finger, automatically unlocking and popping the cabin door, placing the detection card on a sample bracket, then pushing the detection card cabin door to close, inserting the detection card into the leucocyte counting instrument, and placing a detection card detection area in an imaging area of a photoelectric detection module; the user starts the detection process by key operation, the main control unit drives and lights the illumination light source, and then the image sensor is controlled to collect the fluorescence image, as shown in fig. 5.
(4) Image processing and calculation
After the image acquisition is finished, the main control unit performs image processing: all fluorescence spots were first counted, and the number of independent fluorescence spots was taken as the total number of leukocytes directly measured.
Experiment one:
in the experiment, the handheld leucocyte counter is compared with the current clinical examination equipment in hospitals: selecting 36 outpatient blood samples with leukocyte count of 0.3-77.5 × 109And (5) cell/L. The hospital clinical examination equipment is a Sysmex XE-5000 blood cell analyzer of the Hessemcon. The detection method of the handheld leucocyte counting instrument is the same as the second embodiment, and the experimental results are shown in the table 1.
TABLE 1 accuracy evaluation results of the hand-held white blood cell counter of the present invention
From the results in Table 1, it can be seen that the hand-held white blood cell counter of the present invention is at 0.3X 109cell/L-77.5× 109The cell/L has accurate quantitative detection capability and 0.9903 consistency with a large clinical full-automatic blood cell analyzer, and is shown in figure 6.
To sum up, the hand-held white blood cell counter comprises a shell suitable for being held by hand, and a photoelectric detection module, a main control unit, a man-machine interaction module detection card door, a bracket, a detection card and the like which are assembled together, wherein the detection card is in a slit micro-fluidic chip structure and counts white blood cells by a fluorescence excitation principle. The instrument has small volume, light weight and convenient carrying and installation, the counting method can avoid the interference of impurities, has accurate counting, high precision, simple detection operation and high detection efficiency, and is particularly suitable for detecting the leucocytes in primary medical treatment, mobile medical treatment and emergency rescue medical diagnosis in special environments.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the content of the present invention.
Claims (10)
1. A hand-held white blood cell counter is characterized by comprising
The human-computer interaction module is used for inputting a user instruction and outputting a detection result;
the photoelectric detection module is used for detecting and receiving an optical signal emitted by the blood sample dyed by the nucleic acid dyeing agent;
the main control unit is used for transmitting a user instruction input by the human-computer interaction module to the photoelectric detection module, controlling the photoelectric detection module to detect, processing an optical signal received by the photoelectric detection module, calculating the count of white blood cells, and transmitting a detection result of the photoelectric detection module to the human-computer interaction module; and
the detection card is used for bearing the blood sample;
still including being suitable for handheld casing, photoelectric detection module and main control unit all settle inside the casing, and the human-computer interaction module is established at the casing upper surface, and a side of casing is equipped with the detection card hatch door, can take out or put into the casing, and the detection card hatch door is equipped with the bracket that stretches into to the casing inside in the casing, is equipped with on the bracket with detection card size matched with detection card cabin, detects the screens in the photoelectric detection module, and the main control unit is connected with human-computer interaction module and photoelectric detection module electricity.
2. The hand-held leucocyte meter according to claim 1, wherein a lock is provided at the end of the bracket extending into the housing, and a self-locking buckle is provided at the position of the inner wall of the housing corresponding to the lock for locking and ejecting the bracket; preferably, the inner wall of the bottom surface of the shell is provided with a sliding rail matched with the bracket.
3. The hand-held cytometer according to claim 1 or 2, wherein the photodetection module comprises:
a light source for emitting fluorescent light;
a filter for transmitting light of a fixed wavelength range among the light emitted from the light source;
the light splitting sheet is used for reflecting the excited fluorescence of the detection card to the imaging lens;
the imaging lens is used for imaging the fluorescence excited by the detection card;
a fluorescence filter for transmitting light of a fixed wavelength range in the imaging fluorescence; and
the image sensor is used for receiving the optical signal transmitted by the fluorescent filter;
the light splitting sheet is arranged at a position where the fluorescence excited by the detection card can irradiate, the imaging lens is arranged at a position where the fluorescence reflected by the light splitting sheet can be received, and the light source and the image sensor are both electrically connected with the main control unit.
4. The hand-held white blood cell counter according to claim 3, wherein the light source, the optical filter and the light splitter are sequentially arranged at intervals in the photoelectric detection module, the detection card is arranged between the optical filter and the light splitter, the detection card is parallel to the optical filter, and the included angle between the light splitter and the optical filter is 45 °; the light splitting piece, the imaging lens, the fluorescent light filter and the image sensor are sequentially arranged at intervals, the imaging lens, the fluorescent light filter and the image sensor are parallel to each other, the included angle between the detection card and the fluorescent light filter is 90 degrees, and the included angle between the light splitting piece and the fluorescent light filter is 45 degrees; the light emitted by the light source sequentially passes through the optical filter, the detection card, the light splitting sheet, the imaging lens, the fluorescent optical filter and the image sensor.
5. The hand-held white blood cell counter of any one of claims 1-4, wherein the card has a hollow chamber, and an anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the hollow chamber; preferably, the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is one or more of surfactant selected from quaternary ammonium salt, bile acid, glacial acetic acid, saponin, TrionX-100, Tween20, Tween80, NP-40, etc.; the stain is one or more of fluorescent dyes such as SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and the like.
6. The hand-held white blood cell counter of claim 5, wherein the detection card comprises a support substrate and a hollow chamber disposed on the support substrate, the chamber is a sampling detection chamber formed by two parallel chamber sidewalls with a gap therebetween, and has a detection region, a sampling port and a flow guide groove region connecting the sampling port and the detection region, the detection region has a single thickness HDetection ofAnd the thickness of the detection area is less than the thickness H of the flow guide groove areaGuide tube。
7. The hand-held white blood cell counter according to claim 5 or 6, wherein the sampling port is located at an opening of the upper end edge of the two chamber side walls of the sampling and detecting chamber, the upper end edge of one chamber side wall located at the sampling port is provided with a sampling notch, the sampling port is arc-shaped, an included angle α between a tangent line of a downward sliding arc line and a horizontal reference plane of the sampling port determines a flow direction of the liquid sample to be measured entering the flow guide groove area, and the included angle α has a value ranging from 15 ° to 45 °.
8. The hand-held white blood cell counter according to any one of claims 5-7, wherein the detection card further comprises a sample processing chamber disposed on the support substrate, as a storage container for dry or liquid reagents and a mixing operation container for mixing reagents and blood samples, so that the detection card can be further used for pretreatment such as mixing, staining and the like of blood samples.
9. A method for counting leukocytes, which comprises using the handheld leukocyte counter of any one of claims 1-8, and sequentially comprising the steps of preparing a blood sample from whole blood into a sample solution to be tested, adding the sample solution to be tested into a test card for hemolysis and fluorescent staining, placing the test card into the handheld leukocyte counter, fluorescent excitation and detection, image processing and calculation, and the like;
preferably, the fluorescence excitation and detection specifically comprises:
the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the optical filter, the detection card, the light splitting sheet, the imaging lens, the fluorescent optical filter and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit; or
Preferably, the image processing and calculating specifically includes: the main control unit counts all the fluorescence points first, and the number of the independent fluorescence points is used as the total number of the white blood cells.
10. The method for counting leukocytes according to claim 9, wherein the step of adding the sample solution to be tested to the test card comprises: the sampling port is immersed in the sample liquid to be tested for sampling, so that the sample liquid to be tested flows into the detection area from the flow guide groove area under the action of capillary force and is filled with the sample liquid, wherein the capillary force and the thickness of the detection area and the thickness of the flow guide groove area satisfy the following relations:
alternatively, the first and second electrodes may be,
and injecting the sample liquid to be detected into the sampling detection cavity through the sampling notch for sampling, so that the sample liquid to be detected flows into the detection area from the diversion groove area and is filled.
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Cited By (3)
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| CN113222969A (en) * | 2021-05-28 | 2021-08-06 | 大连海事大学 | Imaging flow cytometry adhesion counting and activity detection method and device based on bidirectional background difference method |
| CN114264804A (en) * | 2022-03-03 | 2022-04-01 | 深圳市帝迈生物技术有限公司 | Sample analyzer and detection method thereof |
| WO2023028835A1 (en) * | 2021-08-31 | 2023-03-09 | 深圳迈瑞动物医疗科技股份有限公司 | Specimen analysis apparatus and specimen analysis method |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113222969A (en) * | 2021-05-28 | 2021-08-06 | 大连海事大学 | Imaging flow cytometry adhesion counting and activity detection method and device based on bidirectional background difference method |
| CN113222969B (en) * | 2021-05-28 | 2024-05-14 | 大连海事大学 | Imaging flow type cell adhesion counting and activity detecting method and device based on bidirectional background difference method |
| WO2023028835A1 (en) * | 2021-08-31 | 2023-03-09 | 深圳迈瑞动物医疗科技股份有限公司 | Specimen analysis apparatus and specimen analysis method |
| CN114264804A (en) * | 2022-03-03 | 2022-04-01 | 深圳市帝迈生物技术有限公司 | Sample analyzer and detection method thereof |
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