CN111912767A - White blood cell counting and typing instrument and white blood cell counting and typing method - Google Patents

White blood cell counting and typing instrument and white blood cell counting and typing method Download PDF

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CN111912767A
CN111912767A CN201910385012.3A CN201910385012A CN111912767A CN 111912767 A CN111912767 A CN 111912767A CN 201910385012 A CN201910385012 A CN 201910385012A CN 111912767 A CN111912767 A CN 111912767A
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fluorescent
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light source
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李悦琴
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Suzhou Zhongke Sujing Biotechnology Co.,Ltd.
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Pioneer Precision Beijing Biotechnology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • G01N2015/144—Imaging characterised by its optical setup
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1486—Counting the particles

Abstract

The invention discloses a leucocyte counting and typing instrument and a leucocyte counting and typing method, which comprise the following steps: the device comprises a human-computer interaction module, a photoelectric detection module, a main control unit and a detection card, wherein the detection card is arranged in the photoelectric detection module, and the main control unit is electrically connected with the human-computer interaction module and the photoelectric detection module. The leucocyte counting typing instrument does not need hemolysis or pH value adjustment, has short staining time, simple operation and accurate leucocyte classification. The leucocyte counting and typing method provided by the invention can be used for typing and counting leucocytes, and can also be used for imaging and counting neutrophils, lymphocytes and monocytes.

Description

White blood cell counting and typing instrument and white blood cell counting and typing method
Technical Field
The invention relates to the technical field of blood analysis and detection, in particular to a leucocyte counting and typing instrument and a leucocyte counting and typing method.
Background
Blood cells in the human body are divided into three categories: white blood cells, red blood cells, platelets. Leukocytes mainly include three types, granulocytes, lymphocytes and monocytes. Wherein, the granulocyte accounts for 50-70% of the leukocyte, the lymphocyte accounts for 20-40% of the leukocyte, and the monocyte accounts for 1-7%. Under normal conditions, the total number of leukocytes and the percentage of leukocytes of each type in a human body are relatively stable, and inflammation or other diseases can cause changes in their numbers. That is, the total amount of leukocytes and the percentage of leukocytes of various types are changed when the body is inflamed or otherwise diseased, so that the change of the value can be used as an index of inflammation of the human body, and whether the body is inflamed or not or whether certain diseases are suffered or not can be judged through the change of the value. Therefore, the classification and counting of the white blood cells are important indexes for modern clinical detection and diagnosis.
At present, the methods for classifying and counting leukocytes mainly include a manual microscope method and a blood cell analyzer method.
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 to complete the detection, is long in time consumption, is not suitable for screening a large number of healthy people, and is large in error due to the fact that analysis results are possibly different due to changes of operators.
The coulter method and flow cytometry are also used to determine the number of leukocytes, and the coulter method counts the blood cells based on their nonconductive nature and using the resistance change caused by the suspended blood cells in electrolyte solution as the detection parameter, and because the resistance change is related to the cell size, the different types of leukocytes have different volumes, thus realizing the counting of different types of leukocytes. The flow cytometry method simultaneously utilizes a photoelectric colorimetric principle, a laser scattering principle, a fluorescent staining technology, a sheath flow technology and the like to detect scattered light and fluorescent pulse signals generated by each cell one by one in a flow path, receives the scattered light and fluorescent pulse signals one by one, and realizes the classification and counting of different types of blood cells by calculating the number of the received pulse signals. The blood cell analyzer based on the flow cytometry is the most mainstream examination equipment at present, has the characteristics of high detection speed, high efficiency and capability of avoiding human factor interference, but has the advantages of high price, large volume and high maintenance requirement, is suitable for clinical assay in large hospitals, and is difficult to be suitable for bedside detection, basic diagnosis and treatment of patients in special environments.
In addition, a new kind of leucocyte counting device, hemoCue, adopts the principle of hemolysis counting method: dissolving red blood cells in blood by using a hemolytic agent, dyeing white blood cells by using a cell dyeing agent (such as methylene blue, methyl green or gentian violet and the like) to dye the white blood cells so as to dye the white blood cells, placing a dyed sample under transmitted light, wherein the color of the dyed sample is deepened due to the fact that the white blood cells are dyed, the white blood cells can be represented as dark points on an image under the irradiation of the transmitted light, the total number of the white blood cells can be calculated by calculating the number of the dark points, and different types of white blood cells can be calculated by calculating the form of the dark points. 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 red blood cells is incomplete when the detection time is too short, and the white blood cells are dissolved when the detection time is too long; thirdly, the types of the white blood cells are distinguished through geometric characteristics, and the accuracy is low.
Disclosure of Invention
The present invention aims to overcome the technical defects in the prior art, and provides a leucocyte counting and typing instrument for rapidly detecting the number and the type of leucocytes in blood in a first aspect, which comprises:
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 and finishing counting and typing 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 at least used for bearing a blood sample and is provided with a hollow chamber, and one detection area or a plurality of detection areas are arranged in the chamber;
the detection card is arranged in the photoelectric detection module, and the detection area is positioned in an imaging area of the photoelectric detection module; the main control unit is electrically connected with the man-machine interaction module and the photoelectric detection module.
The photoelectric detection module comprises
A light source for emitting fluorescent light;
a collimating lens for refracting light emitted from the light source into parallel light;
a filter for transmitting light of a fixed wavelength range among the light emitted from the light source;
the fluorescence filter is used for transmitting light in a fixed wavelength range in fluorescence excited by the detection card;
the imaging lens is used for imaging the light transmitted by the fluorescent filter; and
the image sensor is used for receiving images formed by the imaging lens;
the detection card is arranged at the position of the optical filter where the transmitted light can be directly radiated, the fluorescent optical filter is arranged at the position where the fluorescent light excited by the detection card 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, a collimating lens, a light filter, a fluorescent light filter, an imaging lens and an image sensor are arranged in parallel at intervals in sequence, a detection card is positioned between the light filter and the fluorescent light filter and is parallel to the light filter, and light emitted by the light source sequentially passes through the collimating lens, the light filter, the detection card, the fluorescent light filter, the imaging lens and the image sensor.
The photoelectric detection module also comprises a light splitting sheet which is used for reflecting the transmitted light of the optical filter to the detection area of the detection card and transmitting the excited fluorescence of the detection area to the fluorescence optical filter;
preferably, the light source, the collimating lens, the optical filter and the light splitting sheet are arranged at intervals in sequence, the collimating lens is parallel to the optical filter, and an included angle between the light splitting sheet and the optical filter is 45 degrees; the detection card, the light splitting sheet, the fluorescent filter, the imaging lens and the image sensor are sequentially arranged at intervals, the detection card, the fluorescent filter, the imaging lens and the image sensor are mutually parallel, the included angle between the detection card and the light splitting sheet is 90 degrees, and the included angle between the light splitting sheet and the detection card is 45 degrees; the light emitted by the light source sequentially passes through the collimating lens, the optical filter, the light splitting sheet, the detection card, the light splitting sheet, the fluorescent optical filter, the imaging lens and the image sensor.
The photoelectric detection module also comprises a scattered light imaging block which is used for distinguishing lymphocytes and monocytes in the white blood cells;
preferably, the scattered light imaging block comprises a scattered light illumination light source, a scattered light lens and a scattered light filter which are arranged in parallel at intervals in sequence, the detection area of the detection card is arranged at the position, which can be irradiated by the transmitted light of the scattered light filter, the light emitted by the scattered light illumination light source sequentially passes through the scattered light lens and the scattered light filter and irradiates on the detection area of the detection card, and the scattered light illumination light source is electrically connected with the main control unit.
An anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the hollow cavity of the detection card; preferably, the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is selected from surfactant such as triton X-100, quaternary ammonium salt or saponin, etc., and the staining agent is selected from acridine orange fluorescent dye.
The hollow cavity of the detection card 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, a diversion groove area for communicating the sampling opening with the detection area, and the thickness H of the detection areaDetection ofLess than thickness H of flow guide groove areaGuide tube；
Preferably, the sampling port is in a concave arc shape, 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 a flow 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 sampling port is positioned at the upper end edge opening of the two cavity side walls of the sampling detection cavity, and the upper end edge of one cavity side wall positioned at the sampling port is provided with a sampling notch.
The detection regions are two or more, the detection regions are unequal in thickness and independent in space but communicated, the detection regions are used for counting cells with large thickness, and the detection regions are used for parting cells with small thickness.
In a second aspect, the invention provides a leukocyte counting and typing method for rapidly detecting the number and type of leukocytes, which uses the leukocyte counting and typing instrument and sequentially comprises the steps of diluting whole blood 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 leukocyte counting and typing instrument, performing fluorescent excitation and detection, processing and calculating images and the like;
preferably, the fluorescence excitation and detection are specifically performed in three ways:
the first method is as follows: the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit;
the second method comprises the following steps: the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the light splitting sheet, the detection card, the light splitting sheet, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit;
the third method comprises the following steps: the main control unit controls the light source to be turned on and turns off the scattered light illuminating light source, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit; the main control unit controls the scattered light illuminating source to be turned on and turned off, light emitted by the scattered light illuminating source sequentially passes through the scattered light lens, the scattered light optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected scattered light image to the main control unit.
The image processing and calculation specifically comprises the following steps: the main control unit counts all the fluorescent points at first, and the number of the independent fluorescent points is taken as the total number of the white blood cells; then, respectively counting the green light intensity and the red light intensity of all the fluorescent points, wherein the high red light intensity is used as the number of granulocytes in the white blood cells, and the high green light intensity is used as the number of lymphocytes and monocytes;
preferably, the scattered light signal is finally processed, the scattered light signal being relatively weak as the number of lymphocytes and the scattered light signal being relatively strong as the number of monocytes.
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 diversion groove area under the action of capillary force and is filled with the detection area, wherein the capillary force and the thickness of the detection area and the thickness of the diversion groove area satisfy the following relations:
Figure BDA0002054560790000041
alternatively, the first and second electrodes may be,
and injecting the sample liquid to be detected into the sampling detection cavity through the sampling gap for sampling, so that the sample liquid to be detected flows into the detection area from the diversion groove area and is filled.
The thickness of the reaction cavity in the leucocyte counting and typing instrument is 30-100 mu m, the number of the leucocyte overlapping layers can be smaller than 5, the problem that the fluorescence irradiating on the leucocytes at the lower layer cannot be transmitted due to cell overlapping is avoided, and the accuracy of the detection result is improved. The dye in the leucocyte counting and typing instrument is stored in a dry powder state, so that the dye is convenient to store and transport.
The leucocyte counting and typing instrument is simple to use, only fresh blood needs to be added into the reaction cavity, the blood does not need to be diluted or centrifuged, and after the blood is added, the blood is measured after 3 minutes; and the blood may be either blood or venous blood. The leucocyte counting and typing instrument can be used together with a slit microfluidic chip for liquid diversion based on a slit structure, the aim of quickly, automatically and accurately measuring trace liquid is fulfilled, and the typing and the counting of leucocytes in a liquid sample are completed at one time.
The leucocyte counting and typing method of the invention utilizes the fluorescent dye for dyeing nucleic acid to dye leucocytes, and under the excitation of fluorescence, erythrocytes and blood plasma are not influenced by a dyeing agent and have no fluorescence; the combination of the granulocyte and the staining agent excites orange yellow light, and the lymphocyte and monocyte layer emit bright green light. The granulocytes and lymphomonocytes in leukocytes can be classified according to the color of the excited fluorescence. Since 90% of lymphocytes in blood have a diameter of 5-8 μm and monocytes have a diameter of 10-20 μm, lymphocytes and monocytes can be classified according to the size of the cell diameter. The method does not need hemolysis or pH value adjustment, has short dyeing time, simple operation and accurate leukocyte classification. The leucocyte counting and typing method provided by the invention can be used for typing and counting leucocytes, and can also be used for imaging and counting neutrophils, lymphocytes and monocytes.
Drawings
FIG. 1 is a schematic view of a leucocyte count and typing apparatus according to an embodiment;
FIG. 2 is a schematic view of a leukocyte counter-typing apparatus according to an embodiment;
FIG. 3 is a schematic view of a three-chamber white blood cell counter-typing apparatus according to an embodiment;
FIG. 4 is a schematic view showing the structure of the leucocyte count and typing apparatus of the present invention.
FIG. 5 is a flow chart of the method for counting and typing leukocytes according to the present invention;
FIG. 6 is a photograph showing leukocytes after fluorescent staining by the method for counting and typing leukocytes according to the present invention;
FIG. 7 is a red and green fluorescence scattergram obtained by the fluorescence image processing of the present invention;
FIG. 8 is a fluorescent photograph showing the counting and typing of leukocytes by the method of the present invention;
FIG. 9 is a photograph of a scattered light image of a white blood cell count-typed by the method of the present invention;
FIG. 10 is a histogram of a lymph, monocyte scatter image processed using the method of the present invention;
FIGS. 11A-11C are schematic views of a detection card according to an embodiment of the invention;
FIG. 12A is a schematic diagram of a plane structure of a fourth exemplary card according to the present invention;
FIG. 12B is a schematic perspective view of a fourth exemplary card of the present invention;
FIG. 12C is a cross-sectional view taken along line C1-C1 in FIG. 12A.
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 classify and 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. Furthermore, because the granulocytes in the white blood cells emit orange-yellow fluorescence after being dyed, and the lymphocytes and the monocytes emit bright green fluorescence after being dyed, the granulocytes can be distinguished from the lymphocytes and the monocytes by utilizing the principle, and the number of the granulocytes is calculated according to the corresponding cell of each fluorescence point. Finally, the diameters of the lymphocytes and the monocytes are different, the two cells are distinguished by a scattered light principle, the number of the lymphocytes and the monocytes is calculated, and the counting and the typing of the white blood cells are completed at the same time.
On the basis, the invention provides a leucocyte counting and typing instrument, which mainly comprises a human-computer interaction module II, a main control unit 8, a photoelectric detection module I and a detection card 4 as shown in figure 4.
The detection card 4 is at least used for bearing a blood sample and is provided with a hollow chamber, and the inner wall of the hollow chamber is specially designed to be pre-packaged with an anticoagulant, a hemolytic agent and a staining agent; the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is selected from surfactant such as triton X-100, quaternary ammonium salt or saponin, etc., and the staining agent is selected from acridine orange fluorescent dye. The detection card 4 comprises a support substrate and a hollow chamber arranged on the support substrate, the chamber is a sampling detection cavity, is a semi-open cavity formed by two parallel chamber side walls with a certain gap, and is provided with one or more detection areas with different thicknesses, a sampling port and a flow guide groove area communicated with the sampling port and the detection areas, the sampling port is in a concave arc shape, an included angle alpha between a tangent line of a downward sliding arc line and a horizontal reference surface of the sampling port determines the flow direction and 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 optimized detection card is also provided with a sampling notch in the sampling port so as to facilitate liquid transfer and sample introduction.
The human-computer interaction module II mainly comprises a shell 14 of the instrument, a detection card cabin door, a display screen 11 and function keys, wherein the shell 14 is used for protecting internal components of the instrument, the detection card cabin door is used for placing the detection card 4, and the display screen 11 and the function keys finish 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, 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 parting counting into readable results to be fed back to the display screen of the man-machine interaction module.
The photoelectric detection module I comprises a light source 1, a collimating lens 2, an optical filter 3, a fluorescent optical filter 5, an imaging lens 6 and an image sensor 7; a detection card 4 is arranged between the optical filter 3 and the fluorescent optical filter 5; the main control unit is electrically connected with the light source 1 and the image sensor 7. The light source 1 is a fluorescence excitation light source, and the wavelength of the emitted light beam is determined by the fluorescence characteristics of the detected sample, such as the wavelength of the light source 1 is 470-490nm for SYTO-9 stained cells. The light source 1 is arranged at the back focal point of the collimating lens 2 (with the light transmission direction as the front), the focal length of the collimating lens 2 is 10-20mm, and light emitted by the light source 1 forms parallel light beams after passing through the collimating lens 2. The light beam passing through the collimating lens 2 is filtered by the filter 3 and then irradiates on the detection card 4. The filter 3 is a band-pass filter, the transmission wavelength of which is determined by the fluorescence characteristics of the tested sample, such as 470-490nm for SYTO-9 stained cells. The target cells in the detection card 4 emit fluorescence under the excitation action of light beam irradiation, and the fluorescence is focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6 in sequence. The transmission wavelength range of the fluorescence filter 5 is determined by the fluorescence characteristics of the tested sample, such as the transmission wavelength range of the fluorescence filter 5 is 520-650nm for SYTO-9 stained cells; in addition, the fluorescence filter 5 is cut off (blocked) for the light transmitted by the filter 3, the cut-off efficiency is better than 0.01%, that is, the transmittance of the transmitted light via the filter 3 is less than 0.01% when the transmitted light passes through the fluorescence filter 5, because the fluorescence wavelength emitted after the sample is excited by the light is 520 and 650nm, and the light out of this range is filtered by the fluorescence filter 5 to avoid the interference of the light beam before excitation. The object space numerical value space of the imaging lens 6 is larger than 0.1, and the object image magnification is not smaller than 0.5X. The image sensor 7 is an area-array camera with a resolution of more than 100 ten thousand pixels. 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.
Further, the photoelectric detection module can also comprise a scattered light imaging block, because the light intensity of the scattered light is sensitive to the size of the cells, the cells with different sizes can be distinguished by the scattered light. Since the fluorescence of the lymphocytes and the monocytes is green, but the volume of the monocytes is larger than that of the lymphocytes, the lymphocytes and the monocytes can be further distinguished by the scattered light after the scattered light imaging block is added. The scattered light imaging block comprises a scattered light illuminating light source 1 ', a scattered light lens 2 ' and a scattered light filter 3 ' which are arranged in sequence.
The scattered light illuminating source 1 'is a scattered light imaging illuminating source, preferably a light source with a wavelength of more than 600nm, is positioned at a back focal point of the scattered light lens 2' (with the light propagation direction as the front), and a scattered light filter 3 'is arranged in front of the scattered light lens 2' and is used for filtering light with a wavelength of less than 600 nm. The scattered light emitted by the scattered light illumination light source 1 'is converged by the scattered light lens 2' to form parallel light, the parallel light is irradiated on the detection card 4 after impurity light is filtered by the scattered light filter 3 ', lymphocytes and monocytes with different sizes can generate scattered light with different light intensities under the irradiation of the scattered light, the scattered light passing through the scattered light filter 3' is irradiated on the detection card 4 at an incident angle of more than 45 degrees, so that only the scattered light irradiated on the detection card 4 can enter the imaging lens 6, and direct transmitted light irradiated on the detection card 4 cannot enter the imaging lens 6. The scattered light reflected by the detection card 4 is focused and imaged in the image sensor 7 through the fluorescent filter 5 and the imaging lens 6 in sequence.
The scattered light illuminating source 1 'is also connected with the main control unit 8, and the main control unit 8 controls the on or off of the scattered light illuminating source 1'; the light source 1 and the light source 1' are lighted in time-sharing mode and cannot be lighted at the same time; after the main control unit 8 acquires the image signal of the image sensor 7, the fluorescent signal is processed firstly, the number of granulocytes and the total number of lymphocytes and monocytes are calculated, then the scattered light signal is processed, the respective numbers of the lymphocytes and the monocytes are calculated respectively, and then the scattered light signal is transmitted to the man-machine interaction module by the main control unit 8 and is output through the display screen.
The application method of the leucocyte counting and typing instrument comprises the following steps: when the leucocyte counting and typing instrument is used, a user clicks a detection function key on a touch screen, a finger is prompted to press the detection card cabin door 13 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 invention also provides a leucocyte counting and typing method, the flow of which is shown in figure 5, and the method comprises the following steps:
(1) sampling of the sample
Mixing whole blood with diluent (pure water, normal saline or phosphate buffer solution) at a ratio of 1:3 to obtain 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 selected from surfactant such as triton X-100, quaternary ammonium salt or saponin, etc., and the staining agent is selected from acridine orange fluorescent dye;
(3) fluorescence excitation and detection
Inserting the reacted detection card 4 into the leucocyte counting typing instrument, and arranging a detection card detection area 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: counting all fluorescence points, and taking the number of independent fluorescence points as the total number of the white blood cells which are directly measured; then, the green light intensity and the red light intensity of all the fluorescent spots are counted respectively, the number of granulocytes is calculated according to the ratio of the red light intensity, and the number of lymphocytes and monocytes is calculated according to the ratio of the green light intensity.
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. 11A to 11C are structural examples of the detection card 4 of the present invention, which is designed in the form of a slit microfluidic chip. In the first embodiment shown in fig. 11A-11C, the detection card comprises a supporting substrate 21 and a sampling detection cavity 22 disposed on the supporting substrate 21, wherein the supporting substrate 21 is a holding portion of the detection card, which is designed to be suitable for holding, and in the first embodiment, the holding portion is rectangular, and the front end of the holding 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; at least one detection zone 25 may have a single thickness HDetection ofThe thickness is generally in the range of 60 to 120. mu.m. As shown in fig. 11B, the sample enters the detection area 25 to form a detection area 25 with a large thickness, and the detection area has a large sample carrying capacity per unit area and a large depth of field, and is suitable for the overall accurate measurement of the number of cells; the thickness is small, and the spreading area of the liquid sample with the same volume on the detection surface is large, so that the method is suitable for precisely distinguishing cell types.
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. 11C, 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:
Figure BDA0002054560790000091
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:
Figure BDA0002054560790000092
11A-11C, one detection area 25 is disposed in the sampling detection chamber 22 and has a single thickness HDetection ofThe shape is a round-corner rectangle. When the thickness H of the detection zone 25Detection ofWhen the thickness is larger, the depth of field of a detection surface formed on the side wall 24 of the chamber is large, the bearing capacity of a sample in unit area is large, the whole accurate measurement of the number of cells is suitable, and the thickness of the detection area 25 is preferably 90-120 μm; when the thickness H of the detection zone 25Detection ofWhen the thickness is smaller, the depth of field of the detection surface formed on the side wall 24 of the chamber is small, the spreading area of the sample in unit volume is large, the detection surface is suitable for precisely distinguishing cell types, and the thickness of the detection area 25 is preferably 60-90 μm.
Furthermore, one of the two chamber side walls 24 is provided with a sampling notch 29 at the upper end edge of 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 in a concave arc shape, and the included angle α (see fig. 11A) between the tangent of the downward sliding arc line (left arc line in the figure) 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 α is 15 ° to 45 °, 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.
Specifically, the diversion trench region 26 is located in the sampling detection cavity 22 and communicated with the sampling port 27 and the detection region 25, and the thickness H of the diversion trench region 26Guide tubeThickness H greater than detection zone 25Detection ofAvoiding bubbles generated in the sample introduction process as much as possible; in order to ensure that the liquid sample to be measured can continuously flow from the diversion groove area 26 into the detection area 5 and fill the sampling detection area 25 under the action of capillary force, the capillary pressure is required to be greater than zero, and the calculation of the capillary force refers to formula 2).
The structural design of the first embodiment is suitable for cell counting and typing detection with small cell number or low type abundance per unit volume, or application occasions only needing cell counting or cell typing, and according to the application occasions, the detection area 25 with single thickness is selected and the appropriate thickness H is setDetection ofSo as to perform high-precision measurement of the number of cells or high-precision analysis of the type of individual cells.
Of course, in this embodiment, one detection zone 25 may have multiple thicknesses HTest 1，HExamine 2And the thickness values of the detection area 25 are smaller than the thickness of the diversion groove area 26, so that the capillary force for driving the liquid sample to be detected to enter the sampling detection cavity 22, the thickness of the detection area 25 and the thickness H of the diversion groove areaGuide tubeThe relationship of (c) still satisfies the formula 2). The sample can form detection surfaces with different depths of field and different spreading states when entering the detection area 25 with the thickness change, and the precision measurement of cell counting and typing can be considered simultaneously when the data processing is carried out on the cells in the detection areas with different thicknesses.
Example two:
the detection card of the embodiment is the same as the first embodiment, and the staining agent is acridine orange.
The human-computer interaction module II mainly comprises a shell 14 of the instrument, a detection card cabin door, a display screen 11 and function keys, wherein the shell 14 is used for assembling internal components of the instrument and installing the display screen, the function keys and the like, the detection card cabin door is used for placing the detection card 4, and the display screen 11 and the function keys complete 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, 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 acquisition of a sample image, the image processing of the acquired sample image, and the conversion of a digital signal of sample parting counting into a readable result to be fed back to the display screen of the human-computer interaction module.
The photoelectric detection module i, as shown in fig. 1, sequentially includes a light source 1, a collimating lens 2, a filter 3, a fluorescent filter 5, an imaging lens 6, and an image sensor 7 along a light path, that is, the light source 1, the collimating lens 2, the filter 3, the fluorescent filter 5, the imaging lens 6, and the image sensor 7 are sequentially disposed on a straight line; a detection card 4 is arranged between the optical filter 3 and the fluorescent optical filter 5; the main control unit is electrically connected with the light source 1 and the image sensor 7. Wherein the content of the first and second substances,
the light source 1 is a blue LED with a wavelength of 470-490 nm. The light source 1 is arranged at the back focal point of the collimating lens 2 (the light propagation direction is taken as the front), and light emitted by the light source 1 passes through the collimating lens 2 to form parallel light. The parallel light is filtered by the filter 3 and then irradiated on the detection card 4.
The filter 3 is a band-pass filter, and the transmission wavelength thereof is 470-490 nm. The target cells in the detection card 4 emit fluorescence under the excitation action of light beam irradiation, and the fluorescence is focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6 in sequence.
The transmission wavelength range of the fluorescence filter 5 is 520 nm and 650 nm; in addition, the fluorescence filter 5 is cut off (blocked) for the light with the wavelength of 450-500nm, and the cut-off efficiency is better than 0.01%, that is, the transmittance of the light with the wavelength of 450-500nm (the transmitted light wavelength through the filter 3 is in the range) passing through the fluorescence filter 5 is less than 0.01%, because the fluorescence wavelength emitted after the sample is excited by the light is 520-650nm, and the light out of the range is filtered by the fluorescence filter 5 to avoid the interference of the light beam before excitation.
The focal length of the collimating lens 2 is 10-20 mm. The object space numerical value space of the imaging lens 6 is larger than 0.1, and the object image magnification is not smaller than 0.5X. The image sensor 7 is an area-array camera and has a resolution of 300 ten thousand pixels.
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 characteristics and the quantity of cells in the image are calculated, and then the characteristics and the quantity are transmitted to the human-computer interaction module by the main control unit 8 and are output through the display screen.
The counting and typing method of the embodiment comprises the following steps:
(1) sampling of the sample
Mixing whole blood with normal saline according to a ratio of 1:3 to prepare a sample solution to be detected;
(2) and dyeing the mixture
Adding a sample solution to be detected into a detection area of the detection card 4, standing for 2-3 minutes, dissolving a reagent in the detection area of the detection card by the sample solution to be detected, reacting, completing hemolysis of red blood cells in whole blood and leucocyte fluorescent staining under the action of the reagent, wherein an anticoagulant is citrate, and a hemolytic agent is triton X-100 (polyethylene glycol octyl phenyl ether);
(3) fluorescence excitation and detection
Inserting the reacted detection card 4 into the leucocyte counting typing 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 by 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, as shown in fig. 6.
(4) Image processing and calculation
After the image acquisition is finished, the main control unit carries out image processing, firstly, all the fluorescence points are counted, and the number of the independent fluorescence points is used as the total number N of the white blood cells which are directly measured; then, for all the fluorescent spots, the green light intensity and the red light intensity thereof were counted, respectively, and a two-dimensional scattergram was calculated from the green light intensity and the red light intensity, wherein the red light intensity was high as granulocytes, and N1, and the green light intensity was high as lymphocytes and monocytes, and N2, as shown in fig. 7, N1+ N2.
Example three:
the present embodiment is different from the second embodiment only in that the photoelectric detection module is composed of a light source 1, a collimating lens 2, a filter 3, a beam splitter 9, a fluorescence filter 5, an imaging lens 6, an image sensor 7, a control unit 8, and the like, as shown in fig. 2. The light source 1, the collimating lens 2, the optical filter 3 and the light splitting sheet 9 are sequentially arranged on a horizontal straight line along the light propagation direction, the light splitting sheet 9, the fluorescent optical filter 5, the imaging lens 6 and the image sensor 7 are sequentially arranged on another vertical straight line, the horizontal straight line where the light source 1, the collimating lens 2 and the optical filter 3 are located is perpendicular to the vertical straight line where the fluorescent optical filter 5 and the imaging lens 6 are located, the focuses (namely, the feet) of the two straight lines are the positions of the light splitting sheet 9, the included angle between the light splitting sheet 9 and the horizontal straight line is 45 degrees, and the included angle between the light splitting sheet 9 and the vertical straight line is 45 degrees. The detection card 4 is positioned on a vertical straight line where the fluorescent filter 5, the imaging lens 6 and the like are positioned, and is respectively positioned on two sides of the light splitting sheet 9 together with the fluorescent filter 5, so that the light filtered by the light filtering sheet 3 can be reflected to the detection card 4 by the light splitting sheet, and the fluorescence emitted from the detection card 4 can be transmitted to the fluorescent filter 5; the light splitting sheet reflects light emitted by the light source 1 and transmits fluorescence emitted by the detection card, so that the light emitted by the light source 1 can be greatly prevented from directly entering the imaging lens 6, and the interference on fluorescence imaging is reduced. The light source 1 is a blue LED, and the wavelength of emitted light is 470 and 490 nm; the light source 1 is arranged at the rear focus of the collimating lens 2, so that the light emitted from the light source 1 becomes parallel light after passing through the collimating lens 2, the parallel light is filtered by the optical filter 3 and then reaches the light splitting sheet 9, the optical filter 3 is a band-pass optical filter, and the transmission wavelength of the optical filter 3 is 470-490 nm. The spectroscope 9 is a dichroic spectroscope, and has a reflectance of more than 90% with respect to light emitted from the light source 1 and a transmittance of more than 90% with respect to fluorescence emitted from the detection card 4. Then the parallel light reaching the spectroscope 9 irradiates the detection card 4 under the reflection of the spectroscope, the target cell in the detection card 4 emits fluorescence under the excitation action of the light beam irradiation of the light source 1, and the fluorescence is transmitted by the spectroscope 4 and then focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6 in sequence. The transmission wavelength range of the fluorescence filter 5 is 520 nm and 650 nm; in addition, the fluorescence filter 5 is cut off (blocked) for the light with the wavelength of 450-500nm, and the cut-off efficiency is better than 0.01%, that is, the transmittance of the light with the wavelength of 450-500nm (the transmitted light wavelength through the filter 3 is in the range) passing through the fluorescence filter 5 is less than 0.01%, because the fluorescence wavelength emitted after the sample is excited by the light is 520-650nm, and the light out of the range is filtered by the fluorescence filter 5 to avoid the interference of the light beam before excitation. The object space numerical value space of the imaging lens 6 is larger than 0.1, and the object image magnification is not smaller than 0.5X. The image sensor 7 is an area-array camera and has a resolution of 300 ten thousand pixels. 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 characteristics and the quantity of cells in the image are calculated, and then the characteristics and the quantity are transmitted to the human-computer interaction module by the main control unit 8 and are output through the display screen.
The counting and typing method of this embodiment is the same as that of the second embodiment.
Example four:
fig. 12A-12C show another configuration of the test card of the present invention. The structure of the fourth embodiment is a further improvement on the structure of the first embodiment, and is different from the structure of the first embodiment in that:
in this embodiment, there are two detection areas 25, namely a first detection area 51 and a second detection area 52, and the two detection areas are independent of each other (i.e. disposed at an interval) and are communicated with each other, and the two detection areas are communicated with each other by the diversion trench area 26. In this embodiment, the two detection areas are rectangles with combined round corners and right angles, and the thicknesses are respectively HTest 1、HExamine 2And H isTest 1、HExamine 2Not equal. As illustrated in FIG. 12C, the thickness H of the first detection zone 51Test 1The detection surface formed by the liquid sample to be detected in the first detection area 51 has small depth of field and large spreading area of the sample in unit volume, and can be used for precisely distinguishing cell types; thickness H of the second detection zone 52Test 1Large, large depth of field of the detection surface formed by the liquid sample to be detected in the second detection area 52, large sample bearing capacity per unit area, and suitability for the whole of the number of cellsAnd (6) accurately measuring. The two detection regions 51 and 52 with different thicknesses can be provided in this embodiment to simultaneously take account of the precision measurement of cell counting and typing. The two detection zones 51 and 52 are connected by a channel zone 26 of uniform thickness, of course, the thickness H of the two detection zonesTest 1、HExamine 2Are all less than the thickness H of the diversion trench areaGuide tube。
Of course, the detection regions 51 and 52 may have the same thickness, which is smaller than the thickness of the flow guide groove region 26, and the detection results from the detection regions may be analyzed with accuracy and consistency by providing different detection regions.
The other structures of the fourth embodiment are the same as those of the first embodiment, and are not described again.
Example five:
in this embodiment, the detection card, the human-computer interaction module and the main control unit in the first embodiment or the fourth embodiment are the same as those in the second embodiment, the counting and typing of the white blood cells in this embodiment are completed by the fluorescence image and the scattered light image together, and the photoelectric detection module includes a fluorescence imaging block and a scattered light imaging block as shown in fig. 3. Wherein the content of the first and second substances,
the fluorescence imaging block sequentially comprises a light source 1, a collimating lens 2, an optical filter 3, a fluorescence optical filter 5, an imaging lens 6 and an image sensor 7 according to the direction of a light path, wherein the light source 1, the collimating lens 2, the optical filter 3, the fluorescence optical filter 5, the imaging lens 6 and the image sensor 7 are sequentially arranged on a straight line; a detection card 4 is arranged between the optical filter 3 and the fluorescent optical filter 5; the main control unit is electrically connected with the light source 1 and the image sensor 7.
The light source 1 is a blue LED with a wavelength of 470-490 nm. The light source 1 is arranged at the back focal point of the collimating lens 2 (the light propagation direction is taken as the front), and light emitted by the light source 1 passes through the collimating lens 2 to form parallel light. The parallel light is filtered by the filter 3 and then irradiated on the detection card 4. The filter 3 is a band-pass filter, and the transmission wavelength thereof is 470-490 nm. The target cells in the detection card 4 emit fluorescence under the excitation action of light beam irradiation, and the fluorescence is focused and imaged in the image sensor 7 through the fluorescence filter 5 and the imaging lens 6 in sequence. The target wavelength of the fluorescence filter 5 is 520-650nm, namely, the light with the wavelength more than 510nm can be transmitted; in addition, the fluorescence filter 5 is cut off (blocked) for the light with the wavelength of 450-500nm, the cut-off efficiency is better than 0.01%, that is, the transmittance of the light with the wavelength of 450-500nm (the wavelength of the transmitted light through the filter 3 is in the range) passing through the fluorescence filter 5 is less than 0.01%, because the wavelength of the fluorescence and scattered light emitted after the sample is excited by the light is more than 510nm, and the light out of the range is filtered by the fluorescence filter 5 to avoid the interference of the light beam before the excitation. The object space numerical value space of the imaging lens 6 is larger than 0.1, and the object image magnification is not smaller than 0.5X. The image sensor 7 is an area-array camera and has a resolution of 300 ten thousand pixels. 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 characteristics and the quantity of cells in the image are calculated, and then the characteristics and the quantity are transmitted to the human-computer interaction module by the main control unit 8 and are output through the display screen.
The scattered light imaging block comprises a scattered light illuminating light source 1 ', a scattered light lens 2 ' and a scattered light filter 3 ' which are arranged in sequence. The scattered light illuminating light source 1 'is a red light LED, the wavelength of the emitted light is 610nm, the scattered light illuminating light source is positioned at the back focal point of the scattered light lens 2' (the direction of light propagation is taken as the front), and a scattered light filter 3 'is arranged in front of the scattered light lens 2' and used for filtering out light with the wavelength less than 600 nm. The scattered light emitted by the scattered light illumination light source 1 'is converged by the scattered light lens 2' to form parallel light, the parallel light is irradiated on the detection card 4 after impurity light is filtered by the scattered light filter 3 ', lymphocytes and monocytes with different sizes can generate scattered light with different light intensities under the irradiation of the scattered light, the scattered light passing through the scattered light filter 3' is irradiated on the detection card 4 at an incident angle of more than 45 degrees, so that only the scattered light irradiated on the detection card 4 can enter the imaging lens 6, and direct transmitted light irradiated on the detection card 4 cannot enter the imaging lens 6. The scattered light reflected by the detection card 4 is focused and imaged in the image sensor 7 through the fluorescent filter 5 and the imaging lens 6 in sequence.
The scattered light illuminating source 1 'is also connected with the main control unit 8, and the main control unit 8 controls the on or off of the scattered light illuminating source 1'; the light source 1 and the light source 1' are lighted in time-sharing mode and cannot be lighted at the same time; after the main control unit 8 acquires the image signal of the image sensor 7, the fluorescent signal is processed firstly, the number of granulocytes and the total number of lymphocytes and monocytes are calculated, then the scattered light signal is processed, the respective numbers of the lymphocytes and the monocytes are calculated respectively, and then the scattered light signal is transmitted to the man-machine interaction module by the main control unit 8 and is output through the display screen. Fig. 6 and 7 are respectively a fluorescence image and a scattered light image acquired by the image sensor 7. And (3) comprehensively utilizing the fluorescence image and the scattered light image to count the lymphocytes, the monocytes and the granulocytes in a typing way.
The counting and typing method of the embodiment specifically comprises the following steps:
(1) sampling of the sample
Mixing whole blood with diluent according to a ratio of 1:3 to prepare a sample solution to be detected;
(2) and dyeing the mixture
Adding the sample solution to be detected into the sampling detection cavity of the detection card 4, standing for 2-3 minutes, dissolving the reagent in the detection card by the sample solution to be detected, reacting, and completing hemolysis of red blood cells in whole blood and leucocyte fluorescent staining under the action of the reagent;
(3) fluorescence excitation and detection
Inserting the reacted detection card 4 into the leucocyte counting typing instrument, and placing a detection area of the detection card 4 in an imaging area of a photoelectric detection module; a user starts a detection process through key operation, when the detection card of the first embodiment is used, the main control unit 8 drives and lights the light source 1, and then the image sensor 7 is controlled to collect a fluorescence image, as shown in fig. 8; then, the main control unit 8 turns off the light source 1, drives to turn on the scattered light illuminating light source 1', and controls the image sensor 7 to collect a scattered light image, as shown in fig. 9. When the detection card of the fourth embodiment is used, one detection area 51 of the detection card may be located in the irradiation area (or imaging area) of the light source 1 (fluorescent light source), and the other detection area 52 may be located in the irradiation area (or imaging area) of the scattered light illumination light source 1 ', and the main control unit 8 may drive the lighting light source 1 and the scattered light illumination light source 1' at the same time, and control the image sensor 7 to acquire a fluorescent image and a scattered light image.
(4) Image processing and calculation
After the image acquisition is finished, the main control unit carries out image processing, firstly, all the fluorescence points are counted, and the number of the independent fluorescence points is used as the total number N of the white blood cells which are directly measured; then, for all the fluorescent dots, the green light intensity and the red light intensity thereof were counted, respectively, and a two-dimensional scattergram was calculated from the green light intensity and the red light intensity, wherein the red light intensity was high as granulocytes, counted as N1, and the other part was lymphocytes and monocytes N2, as shown in fig. 7, N ═ N1+ N2. For the lymphocytes and monocytes determined, the scatter intensities were counted in combination with the scatter image, and the histogram distribution is shown in fig. 10, where the left side of the segmentation line (weak scatter light) in the graph is lymphocytes, the number is N21, the right side (scatter intensity) is monocytes, the number is N22, and N2 is N21+ N22.
Experiment:
in the experiment, the leucocyte counting and typing instrument 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 leucocyte counting typing instrument is the same as the second embodiment, and the experimental results are shown in the table 1.
TABLE 1 evaluation results of the accuracy of the leucocyte count typing instrument of the present invention
Figure BDA0002054560790000151
As is clear from the results in Table 1, the leucocyte count typing instrument of the present invention was set to 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.
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 (12)

1. A white blood cell count and typing instrument, 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 and finishing counting and typing 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 at least used for bearing a blood sample and is provided with a hollow chamber, and one detection area or a plurality of detection areas are arranged in the chamber;
the detection card is arranged in the photoelectric detection module, and the detection area is positioned in an imaging area of the photoelectric detection module; the main control unit is electrically connected with the man-machine interaction module and the photoelectric detection module.
2. The apparatus of claim 1, wherein the photodetector module comprises a light source for emitting fluorescence;
a collimating lens for refracting light emitted from the light source into parallel light;
a filter for transmitting light of a fixed wavelength range among the light emitted from the light source;
the fluorescence filter is used for transmitting light in a fixed wavelength range in fluorescence excited by the detection card;
the imaging lens is used for imaging the light transmitted by the fluorescent filter; and
the image sensor is used for receiving images formed by the imaging lens;
the detection card is arranged at the position of the optical filter where the transmitted light can be directly radiated, the fluorescent optical filter is arranged at the position where the fluorescent light excited by the detection card can be received, and the light source and the image sensor are both electrically connected with the main control unit.
3. The apparatus according to claim 2, wherein the light source, the collimating lens, the optical filter, the fluorescence filter, the imaging lens and the image sensor are disposed in parallel and spaced in sequence in the photoelectric detection module, the detection card is disposed between the optical filter and the fluorescence filter and is parallel to the optical filter, and the light emitted from the light source passes through the collimating lens, the optical filter, the detection card, the fluorescence filter, the imaging lens and the image sensor in sequence.
4. The apparatus according to claim 2, wherein the photoelectric detection module further comprises a spectroscope for reflecting the transmitted light from the optical filter to the detection area of the detection card and transmitting the excited fluorescence from the detection area to the fluorescence filter;
preferably, the light source, the collimating lens, the optical filter and the light splitting sheet are arranged at intervals in sequence, the collimating lens is parallel to the optical filter, and an included angle between the light splitting sheet and the optical filter is 45 degrees; the detection card, the light splitting sheet, the fluorescent filter, the imaging lens and the image sensor are sequentially arranged at intervals, the detection card, the fluorescent filter, the imaging lens and the image sensor are mutually parallel, the included angle between the detection card and the light splitting sheet is 90 degrees, and the included angle between the light splitting sheet and the detection card is 45 degrees; the light emitted by the light source sequentially passes through the collimating lens, the optical filter, the light splitting sheet, the detection card, the light splitting sheet, the fluorescent optical filter, the imaging lens and the image sensor.
5. The apparatus of claim 3, wherein the photodetection module further comprises a scattered light imaging block for differentiating between lymphocytes and monocytes in the white blood cells;
preferably, the scattered light imaging block comprises a scattered light illumination light source, a scattered light lens and a scattered light filter which are arranged in parallel at intervals in sequence, the detection area of the detection card is arranged at the position, which can be irradiated by the transmitted light of the scattered light filter, the light emitted by the scattered light illumination light source sequentially passes through the scattered light lens and the scattered light filter and irradiates on the detection area of the detection card, and the scattered light illumination light source is electrically connected with the main control unit.
6. The apparatus according to any one of claims 1-5, wherein an anticoagulant, a hemolytic agent and a staining agent are attached to the inner wall of the hollow chamber of the detection card; preferably, the anticoagulant is one or more of edetate, citrate, oxalate, heparin and the like; the hemolytic agent is selected from surfactant such as triton X-100, quaternary ammonium salt or saponin, etc., and the staining agent is selected from acridine orange fluorescent dye.
7. The leucocyte count and typing instrument according to claim 6, wherein the hollow chamber of the detection card is a sampling and detection chamber, which is a semi-open cavity formed by two parallel chamber side walls with a gap, and has a detection area, a sampling port and a flow guide groove area communicating the sampling port and the detection area, and the thickness H of the detection area isDetection ofLess than thickness H of flow guide groove areaGuide tube；
Preferably, the sampling port is in a concave arc shape, 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 a flow 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.
8. The apparatus according to claim 6 or 7, wherein the sampling opening is located at the opening of the upper edge of the two chamber side walls of the sampling and detecting chamber, and the upper edge of one chamber side wall located at the sampling opening is provided with a sampling notch.
9. The apparatus of claim 6, 7 or 8, wherein the number of the detection regions is two or more, and the detection regions have different thicknesses, are independent and connected, and have a larger thickness for cell counting and a smaller thickness for cell typing.
10. A method for counting and typing leukocytes, which comprises diluting 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 apparatus, exciting and detecting fluorescence, processing and calculating images, etc.;
preferably, the fluorescence excitation and detection are specifically performed in three ways:
the first method is as follows: the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit;
the second method comprises the following steps: the main control unit controls the light source to be started, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the light splitting sheet, the detection card, the light splitting sheet, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit;
the third method comprises the following steps: the main control unit controls the light source to be turned on and turns off the scattered light illuminating light source, light emitted by the light source sequentially passes through the collimating lens, the optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected fluorescent image to the main control unit; the main control unit controls the scattered light illuminating source to be turned on and turned off, light emitted by the scattered light illuminating source sequentially passes through the scattered light lens, the scattered light optical filter, the detection card, the fluorescent optical filter, the imaging lens and the image sensor, and the image sensor transmits the collected scattered light image to the main control unit.
11. The method according to claim 10, wherein the image processing and calculating is specifically: the main control unit counts all the fluorescent points at first, and the number of the independent fluorescent points is taken as the total number of the white blood cells; then, respectively counting the green light intensity and the red light intensity of all the fluorescent points, wherein the high red light intensity is used as the number of granulocytes in the white blood cells, and the high green light intensity is used as the number of lymphocytes and monocytes;
preferably, the scattered light signal is finally processed, the scattered light signal being relatively weak as the number of lymphocytes and the scattered light signal being relatively strong as the number of monocytes.
12. The method according to claim 10 or 11, 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 diversion groove area under the action of capillary force and is filled with the detection area, wherein the capillary force and the thickness of the detection area and the thickness of the diversion groove area satisfy the following relations:
Figure FDA0002054560780000031
alternatively, the first and second electrodes may be,
and injecting the sample liquid to be detected into the sampling detection cavity through the sampling gap for sampling, so that the sample liquid to be detected flows into the detection area from the diversion groove area and is filled.
CN201910385012.3A 2019-05-09 2019-05-09 White blood cell counting and typing instrument and white blood cell counting and typing method Pending CN111912767A (en)

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