WO2021042307A1 - 血液检测方法及血液分析系统 - Google Patents

血液检测方法及血液分析系统 Download PDF

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WO2021042307A1
WO2021042307A1 PCT/CN2019/104451 CN2019104451W WO2021042307A1 WO 2021042307 A1 WO2021042307 A1 WO 2021042307A1 CN 2019104451 W CN2019104451 W CN 2019104451W WO 2021042307 A1 WO2021042307 A1 WO 2021042307A1
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sample
tested
blood
optical information
information
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PCT/CN2019/104451
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English (en)
French (fr)
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易秋实
吴舒晨
汪东生
陈庚文
朱轲
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深圳迈瑞生物医疗电子股份有限公司
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Priority to CN201980099668.7A priority Critical patent/CN114270167A/zh
Priority to PCT/CN2019/104451 priority patent/WO2021042307A1/zh
Publication of WO2021042307A1 publication Critical patent/WO2021042307A1/zh

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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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • 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—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/018—Platelets
    • 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
    • G01N2015/1019—Associating Coulter-counter and optical flow cytometer [OFC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the present invention relates to blood detection, in particular to an optical detection method of platelets and a blood analysis system thereof.
  • Human blood contains various cells such as red blood cells, white blood cells, and platelets. Platelets are non-nucleated cells with a diameter of 2-3 microns. Normal human blood contains 150,000 to 350,000 platelets per microliter.
  • the methods of measuring platelets in blood samples with blood analyzers usually include impedance method and optical method.
  • the impedance method is based on the Coulter principle.
  • the particles in the diluted blood sample are passed through the small hole one by one, and a constant current source is applied on both sides of the small hole.
  • Each cell passing through the small hole causes the electrical impedance of the liquid in the small hole. Change to generate electrical pulses, and then plot the detected electrical pulses into a histogram for analysis.
  • the volume of platelets is the smallest, the volume of white blood cells is the largest, and the volume of red blood cells is in the middle.
  • the detected pulse intensity is related to the volume of the cells passing through the small hole, so through volume division, different cell types can be distinguished.
  • testing some special samples (such as samples containing larger platelets and smaller red blood cells) will affect the accuracy and precision of platelet detection.
  • the optical method is based on flow cytometry, in which the diluted and stained sample is squeezed by the sheath flow so that the cell particles in the sample pass through the optical detection zone in sequence. Each cell is irradiated by the excitation light source, and the forward scatter signal representing the cell volume information and the fluorescent signal of the particles dyed by the fluorescent dye are obtained in the optical detection device to generate a two-dimensional scatter of the forward scatter signal and the fluorescent signal. Dot graph, and then divide and count RBC and PLT.
  • Flow cytometry can quickly determine cells in the blood.
  • US patents US 6,114,173, US 4,882,284, and US 5,891,731 all disclose methods for staining blood cells with dyes to better distinguish platelets under non-hemolytic conditions.
  • Chinese patent application CN 101173921 discloses a specific stain for distinguishing platelets.
  • the optical method can obtain accurate PLT measurement results, it is necessary to add a special detection channel to the blood cell analyzer, which leads to a longer measurement time for a single sample and a slower test speed.
  • this special detection channel also requires special diluents and dyes. Therefore, the equipment cost and detection cost are high, which is not conducive to clinical promotion.
  • the purpose of the present invention is to provide a detection method for detecting platelets in a blood sample without a separate detection channel.
  • the method uses a hemolysis channel, especially a conventional white blood cell detection channel and a light source of a specific wavelength. According to the optical information, the platelets in the blood sample can be accurately identified from the ghost particles, which are usually considered as interference signals.
  • a further object of the present invention is to alarm or identify and count the reticulocyte samples with abnormal content of reticulocytes based on optical information, especially fluorescence information.
  • Another purpose of the present invention is to compare and correct the platelet count detected by the present invention with the platelet count detected by the conventional impedance method.
  • the object of the present invention is to provide a blood detection system implementing the above method.
  • the first aspect of the present invention first provides a blood testing method, the method comprising:
  • the blood sample is treated with a first reagent to obtain a sample to be tested.
  • the first reagent includes a hemolytic agent that lyses the red blood cells in the blood sample into fragments and causes the white blood cells and platelets in the blood sample to become The cell morphology remains basically intact;
  • the particles in the sample to be tested are passed through the detection area of the optical detection device one by one, and the light source of the optical detection device is used to irradiate the particles in the sample to be tested to obtain the optical of the sample to be tested.
  • Information wherein the light source is configured to emit light with a wavelength less than 488nm or emit violet or blue light;
  • the optical information of the platelets in the sample to be tested is obtained according to at least two kinds of light intensity information in the optical information of the sample to be tested.
  • the second aspect of the present invention provides a blood testing method, the method comprising:
  • the blood sample is treated with a first reagent and a second reagent to obtain a sample to be tested.
  • the first reagent includes a hemolytic agent that lyses the red blood cells in the blood sample into fragments and makes the blood sample.
  • the cell morphology of white blood cells and platelets is basically kept intact, and the second reagent includes a fluorescent dye;
  • the particles in the sample to be tested are passed through the detection area of the optical detection device one by one, and the light source of the optical detection device is used to irradiate the particles in the sample to be tested to obtain the optical of the sample to be tested.
  • Information wherein the light source is configured to emit light with a wavelength less than 488nm or emit violet or blue light;
  • the optical information of the reticulocytes in the sample to be tested is obtained according to the fluorescence intensity information and the scattered light intensity information in the optical information of the sample to be tested.
  • a third aspect of the present invention provides a blood detection method, the method comprising:
  • the second sample solution to be tested is treated with a first reagent, and the first reagent includes a hemolytic agent that at least completely lyses mature red blood cells in the second sample solution to be tested into fragments and causes the first 2.
  • the cell morphology of leukocytes and platelets in the sample solution to be tested remains basically intact;
  • the particles in the second sample solution to be tested are passed through the detection area of the optical detection device one by one, and the light source of the optical detection device is used to irradiate the particles in the second sample solution to be tested to obtain the second sample solution.
  • a fourth aspect of the present invention provides a blood analysis system, the blood analysis system comprising:
  • a sampling device having a pipette with a pipette nozzle and a driving device for driving the pipette to quantitatively suck blood samples through the pipette nozzle;
  • the sample preparation device has a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the blood sample drawn by the sampling device, and the reagent supply part provides the first reagent to the reaction tank, so that the sampling
  • the blood sample drawn by the device is mixed with the first reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested, wherein the first reagent includes a hemolytic agent, and the hemolytic agent will Lysing the red blood cells in the blood sample into fragments and keeping the cell morphology of the white blood cells and platelets in the blood sample basically intact;
  • the optical detection device includes a light source, a flow chamber and at least two types of detectors.
  • the particles of the sample to be tested can flow in the flow chamber, and the light emitted by the light source irradiates the particles in the flow chamber to produce optical Information, the detector is used to collect the optical information, wherein the light source is configured to emit light with a wavelength less than 488 nm or emit violet or blue light; and
  • a data processing device that is electrically connected to the optical detection device and includes a processor and a computer-readable storage medium storing a computer program, wherein the data processing device is configured to, when the computer program is executed by the processor, The following steps are performed: obtaining the optical information of platelets in the sample to be tested according to at least two kinds of light intensity information in the optical information of the sample to be tested.
  • a fifth aspect of the present invention provides a blood analysis system, which includes:
  • a sampling device having a pipette with a pipette nozzle and a driving device for driving the pipette to quantitatively suck blood samples through the pipette nozzle;
  • the sample preparation device has a reaction cell and a reagent supply part, wherein the reaction cell is used to receive the blood sample sucked by the sampling device, and the reagent supply part provides the first reagent and the second reagent to the reaction cell, thereby
  • the blood sample drawn by the sampling device is mixed with the first reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested, wherein the first reagent includes a hemolytic agent, and The hemolytic agent lyses the red blood cells in the blood sample into fragments and keeps the cell morphology of the white blood cells and platelets in the blood sample substantially intact, and the second reagent includes a fluorescent dye;
  • the optical detection device includes a light source, a flow chamber, a scattered light detector, and a fluorescence detector.
  • the particles of the sample to be tested can flow in the flow chamber, and the light emitted by the light source illuminates the particles in the flow chamber
  • the scattered light detector is used to collect scattered light intensity information in the optical information
  • the fluorescence detector is used to collect fluorescence intensity information in the optical information, wherein the light source is configured to emit less than 488nm light or emits violet or blue light;
  • a data processing device that is electrically connected to the optical detection device and includes a processor and a computer-readable storage medium storing a computer program, wherein the data processing device is configured to, when the computer program is executed by the processor, The following steps are performed: obtaining the optical information of the reticulocytes in the sample to be tested according to the fluorescence intensity information and the scattered light intensity information in the optical information of the sample to be tested.
  • the present invention provides a new method for detecting platelets, which can obtain accurate platelet information without adding additional detection channels or using additional specific detection reagents.
  • the method uses a light source emitting light with a wavelength below 488 nm, and uses a hemolysis detection channel to detect hemolysis-treated blood samples, so that platelets can be completely distinguished from ghost particles through optical detection.
  • this method can also obtain the analysis results of white blood cells at the same time, and further obtain the detection information of reticulocytes when fluorescent dyes are used, so that the detection information of white blood cells and even reticulocytes can be obtained at the same time in the same detection channel, thereby simplifying
  • the blood test is improved, and the test cost is reduced.
  • Fig. 1 is a schematic flowchart of a blood detection method according to the first aspect of the present invention
  • Figure 2 is a two-dimensional scatter diagram of forward scattered light and side scattered light obtained by processing a blood sample to be tested with a conventional hemolytic agent;
  • Fig. 3 is a schematic flow chart of the blood detection method according to the first embodiment of the first aspect of the present invention.
  • FIG. 5 is a schematic flowchart of a blood testing method according to an example of the second embodiment of the first aspect of the present invention.
  • 6A and 6B are respectively two-dimensional scatter plots obtained according to an example of the second embodiment of the first aspect of the present invention.
  • FIG. 7 is a schematic flowchart of a blood testing method according to another example of the second embodiment of the first aspect of the present invention.
  • 8A and 8B are respectively two-dimensional scatter plots obtained according to another example of the second embodiment of the first aspect of the present invention.
  • Figure 9 is a correlation diagram of the platelet count value PLT-1 measured according to the method of the present invention and the platelet count value PLT-0 measured by a single RET channel;
  • Fig. 10 is a comparison diagram of the linear relationship between the relative count value RET-1 of reticulocytes measured according to the method of the present invention and the count value RET-0 of reticulocytes measured by a separate RET channel;
  • FIG. 11 is a schematic diagram 1 of the composition structure of an optical detection device provided by an embodiment of the present invention.
  • Figure 13 is a schematic flowchart of a blood detection method according to the second aspect of the present invention.
  • Figure 14 is a schematic flow chart of a blood testing method according to the third aspect of the present invention.
  • 15 is a schematic diagram of the composition structure of a blood cell analysis system provided by an embodiment of the present invention.
  • 16 is a schematic diagram of an optical detection device in a blood cell analysis system provided by an embodiment of the present invention.
  • FIG. 17 is a schematic diagram of an impedance detection device in a blood cell analysis system provided by an embodiment of the present invention.
  • the terms "including”, “including” or any other variants thereof are intended to cover non-exclusive inclusion, so that a method or device including a series of elements not only includes what is clearly stated Elements, and also include other elements not explicitly listed, or elements inherent to the implementation of the method or device. Without more restrictions, the element defined by the sentence “including a" does not exclude the existence of other related elements in the method or device that includes the element (such as steps in the method or units in the device).
  • the unit here can be a part of a circuit, a part of a processor, a part of a program or software, etc.).
  • first ⁇ second ⁇ third involved in the embodiment of the present invention only distinguishes similar objects, and does not represent a specific order of objects. Understandably, “first ⁇ second ⁇ third” “Three” can be interchanged in specific order or precedence when permitted. It should be understood that the objects distinguished by “first ⁇ second ⁇ third” can be interchanged under appropriate circumstances, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein.
  • the electrical impedance method is routinely used.
  • the electrical impedance method is not accurate enough for detecting platelets when detecting some special blood samples. For this reason, it has been reported to detect platelets in a separate detection channel using optical detection methods combined with specific detection reagents. However, these methods increase the cost of testing equipment and also increase testing costs.
  • U.S. Patent 7,344,890 B2 discloses a ghosting reagent (ghosting reagent) to process blood samples containing interfering substances to change the scattering characteristics of red blood cells, so that the intensity and flight of the forward scattered light of the cells in the sample can be measured. Over time, platelets are clearly distinguished from red blood cells in the resulting two-dimensional scatter plot. This method significantly changes the refractive index of red blood cells by causing normal red blood cells to lose heme. However, this method cannot effectively distinguish large platelets from white blood cells. Moreover, this method needs to measure the flight time, and platelets cannot be detected only by optical information.
  • the present invention proposes a method for detecting platelets by an optical method under the premise of hemolysis processing of blood samples.
  • the method can use optical information to completely distinguish platelets from red blood cells lysed by hemolysis processing, and can obtain both at the same time.
  • the optical information of reticulocytes can be further obtained, so as to indicate the presence of reticulocytes in the blood sample.
  • a light source emitting purple or blue light or green light or yellow light or a light source emitting light with a wavelength of less than 600 nanometers, especially less than 488 nanometers is used for detection, so as to detect Platelets are completely distinguished from other particles in the ghost zone, so that accurate platelet counts can be obtained.
  • the blood detection method includes the following steps.
  • the blood sample is processed with the first reagent to obtain the sample to be tested.
  • the first reagent includes a hemolytic agent to lyse the red blood cells in the blood sample into fragments, and the platelets can basically maintain their cell morphology, and preferably the white blood cells can also basically maintain their cell morphology.
  • step S12 the optical information of each particle in the sample to be tested is obtained by the optical detection device. Specifically, the particles in the sample to be tested are passed through the detection area of the optical detection device one by one, and the light source of the optical detection device is used to irradiate the particles in the sample to be tested to obtain the Kind of optical information.
  • step S13 the optical information of platelets in the sample to be tested is obtained according to at least two kinds of light intensity information in the optical information of the sample to be tested.
  • the wavelength range of violet light is approximately 370nm-435nm
  • the wavelength range of blue light is approximately 436nm-500nm
  • the wavelength range of green light is approximately 501nm-560nm
  • the wavelength range of yellow light is approximately 561nm-599nm.
  • the light source of the detection system of the present invention allows emission of light with a wavelength approximately in the range of 370 nm to 435 nm, preferably in the range of 375 nm to 420 nm, more preferably in the range of 400 nm to 410 nm.
  • the light source of the detection system of the present invention is allowed to emit light with a wavelength approximately in the range of 436 nm to 500 nm, preferably in the range of 440 nm to 500 nm, more preferably in the range of 445 nm to 490 nm.
  • the light source of the detection system of the present invention is allowed to emit light with a wavelength approximately in the range of 501 nm to 560 nm, preferably in the range of 510 nm to 550 nm, more preferably in the range of 510 nm to 530 nm.
  • the light source of the detection system of the present invention can be configured to emit light with a wavelength of about 375 nm or 405 nm or 450 nm or 520 nm.
  • the method according to the first aspect of the present invention is implemented in a conventional white blood cell classification and/or counting channel, and does not affect the classification and/or counting of white blood cells.
  • the white blood cell area is significantly away from the platelet area. Therefore, the method of the present invention will not cause white blood cells to interfere with large-sized platelets and platelet aggregates. Therefore, the method of the present invention can simultaneously obtain at least three categories (e.g., monocytes, lymphocytes, and neutrophils), and even four categories (e.g., lymphocytes, monocytes, neutrophils) can be obtained when fluorescent dyes are used.
  • the white blood cell test results of neutrophils and eosinophils can be used to alarm or count reticulocytes.
  • the light source used in the blood detection method according to the first aspect of the present invention may be configured to emit light with a wavelength in the range of about 375 nm to 480 nm, especially light in the range of 405 nm to 480 nm, more preferably light in the range of 440 nm to 480 nm. Light.
  • the first reagent may contain a strong hemolytic agent capable of deeply lysing red blood cells, completely lysing the red blood cells in the blood sample into fragments whose light scattering properties are significantly different from those of platelets.
  • the strong hemolytic agent is not particularly limited.
  • such strong hemolytic agents may be alkyl glycosides, triterpene saponins, steroidal saponins, and the like.
  • a specific strong hemolytic agent may be a glycoside compound having the general formula I:
  • R is selected from the group consisting of monosaccharides, deoxymonosaccharides and polysaccharides, and n is an integer of 5-17.
  • glycoside compounds can quickly dissolve red blood cells.
  • Glycoside compounds are compounds formed by dehydration of the hemiacetal hydroxyl groups of sugars (or polysaccharides) with the hydroxyl groups of alkanols.
  • the glycoside compound in the hemolytic agent of the present invention may be a single compound or a mixture of two or more glycoside compounds that conform to the above-mentioned general formula.
  • the concentration of the glycoside compound of the general formula I in the hemolytic agent of the present invention varies according to the nature of the selected glycoside, the reaction time, the reaction temperature and the usage amount of other ingredients, and the usual dosage is 0.025g/L ⁇ 10g /L, preferably 0.1 g/L to 5.0 g/L.
  • the first reagent may include a nonionic surfactant having the general formula II:
  • R 1 is a C8-C23 alkyl group
  • R 2 is -O-, Or -COO-
  • m is an integer of 10-50
  • At least one organic acid or salt thereof wherein the organic acid or salt thereof is selected from the group consisting of organic acids having at least one carboxyl group or sulfonic acid group and alkali metal salts thereof.
  • the non-ionic surfactant of general formula II can bind to the cell membrane to a certain extent, and protect the cell membrane of leukocytes and platelets from the aforementioned glycoside compounds and maintain or substantially maintain their cell morphology.
  • the combination of the compounds of formula I and formula II can achieve the effect of rapid and deep lysis of red blood cells on the one hand, and on the other hand, in order to effectively detect platelets, it can protect the cell membrane of platelets.
  • the amount ratio between the two is also different.
  • the dosage ratio of the compounds of the general formula I and the general formula II is 1:100 to 1:3, preferably 1:25 to 1:5, more preferably 1:10 to 1:5.
  • the first reagent may further include at least one organic acid or a salt thereof to make the white blood cell side scattered light more distinguishable.
  • the first agent of the present invention may further include conventional additives. These additives can be optionally added as needed, such as (but not limited to) buffers, metal chelating agents, osmotic pressure regulators, preservatives and the like. These reagents are all commonly used reagents in the field, as long as the above-mentioned components in the hemolytic agent of the present invention are not prevented from functioning.
  • the mixing ratio of the first reagent and the blood sample according to the first embodiment is not particularly limited.
  • the volume mixing ratio of the blood sample and the first reagent may be 1:40 to 1:60.
  • the hemolysis reaction is performed at a temperature of, for example, 40 to 60°C for 15 to 100 seconds, preferably 40 to 80 seconds. The reaction temperature and time can be adjusted according to specific conditions.
  • deep lysis of red blood cells means that red blood cells are completely lysed into fragments whose light scattering properties are significantly different from those of platelets, and platelets can basically maintain their cell morphology, preferably white blood cells can also basically maintain their cell morphology.
  • platelets and deeply lysed red blood cell fragments can form two completely separated particle groups.
  • routine lysis of red blood cells in this article refers to the use of conventional hemolytic agents. After the hemolytic agent reacts with the blood sample, there may be overlap between the lysed red blood cell fragments and the platelet particle group on the scatter diagram composed of scattered light signals. as shown in picture 2.
  • Fig. 2 only shows the ghost area. It can be seen from Fig. 2 that the reticulocyte fragment area and the platelet area overlap each other to a certain extent, and it is difficult to obtain an accurate count of the platelets.
  • the strong hemolytic agent in particular, can completely lyse the possibly existing reticulocytes into smaller pieces with light scattering characteristics significantly different from platelets. While using conventional hemolytic agents to treat blood cells, although white blood cells and platelets remain roughly intact cell morphology and mature red blood cells are completely lysed into fragments, reticulocytes are partially broken due to their immaturity, and the volume of some reticulocyte fragments may be the same Platelets are similar, so there may be overlap between reticulocyte fragments and platelet particle clusters on the scatter plot composed of scattered light signals.
  • a scatter diagram composed of at least two kinds of scattered light intensity information obtained by optical detection can be clearly distinguished Separate the red blood cell fragment area and the platelet area to realize accurate and precise identification and counting of platelets.
  • a subpopulation of white blood cells including at least monocytes, lymphocytes and neutrophils can be obtained.
  • Fig. 3 shows a schematic flow chart of a specific blood detection method in the first embodiment.
  • the blood sample is processed with the first reagent containing a strong hemolytic agent as described above to obtain the sample to be tested.
  • the particles in the sample to be tested are passed through the flow cell of the optical detection device one by one, so as to obtain optical information.
  • the platelets are distinguished from other particles, especially completely lysed red blood cell fragments, based on the forward direction and at least one other angle of scattered light intensity information.
  • step S114 the optical information of the leukocytes in the sample to be tested is obtained according to the forward scattered light intensity information and the side scattered light intensity information in the optical information of the sample to be tested, so as to obtain the optical information of the leukocytes in the sample to be tested according to the obtained leukocytes.
  • the optical information distinguishes white blood cell subpopulations to obtain at least a subpopulation of white blood cells including monocytes, lymphocytes, and neutrophils.
  • the blood sample is processed by decyl glucoside to obtain the sample to be tested, and then the blood cell analyzer (Mindray BC6200) is used for the measurement.
  • the optical detection device has been modified to set the excitation wavelength of the laser light source to 405nm.
  • Fig. 4 only shows the ghost area. It can be seen from Fig. 4 that the red blood cell fragment area and the platelet area are more distinguishable, and the platelets can be clearly separated from the ghost area for effective statistics.
  • the scattered light intensity information at other angles includes at least one of side scattered light intensity information, middle-angle scattered light intensity information, and high-angle scattered light intensity information.
  • the at least two kinds of scattered light intensity information include forward scattered light intensity information and side scattered light intensity information.
  • the scattering angle of the forward scattered light signal may be about 1°-10°.
  • the scattering angle of the medium-angle scattered light signal may be about 10°-20°.
  • the scattering angle of the high-angle scattered light signal may be about 20° to 70°.
  • the scattering angle of the side scattered light signal may be about 70° to 110°.
  • FIG. 5 shows a flowchart of a specific blood detection method in the second embodiment.
  • the hemolytic agent used in the first reagent of the present invention is not particularly limited. It may be a strong hemolytic agent as described above, or it may only routinely lyse red blood cells into Hemolytic agent for fragments.
  • the blood sample treated with the first reagent is also treated with a second reagent containing at least one fluorescent dye. Referring to FIG. 5, in step S121, the blood sample is processed together with a first reagent containing a hemolytic agent and a second reagent containing a fluorescent dye to obtain a sample to be tested.
  • the first reagent may contain any hemolytic agent as long as it can lyse red blood cells, and the degree of hemolysis is not particularly limited, and may be a strong hemolytic agent as described above. , It can also be a conventional hemolytic agent. Exemplary conventional hemolytic agents such as quaternary ammonium salt cationic surfactants (such as tetradecyl trimethyl ammonium chloride), but the present invention is not limited thereto.
  • the time of the hemolysis reaction can be set shorter, for example, in the range of 15 to 30 seconds, especially about 20 seconds. , In order to speed up the sample detection speed.
  • step S122 the optical information of the sample to be tested is also obtained by the optical detection device.
  • fluorescence intensity information is further obtained.
  • step S123 the optical information of the platelets in the sample to be tested is obtained according to the fluorescence intensity information and the scattered light intensity information in the optical information of the sample to be tested, especially the forward scattered light intensity information, so as to obtain the optical information of the platelets in the sample to be tested.
  • Platelets are distinguished from other particles.
  • step S124 it is also possible to distinguish white blood cell subgroups according to the side scattered light intensity and fluorescence intensity information in the optical information of the sample to be tested to obtain leukocytes including at least monocytes, lymphocytes, and neutral cells. Subpopulations of granulocytes and/or identify immature granulocytes.
  • the optical information of the reticulocytes in the sample to be tested can also be obtained according to the fluorescence intensity information and the scattered light intensity information in the optical information of the sample to be tested.
  • a prompt may also be output that the reticulocytes are present in the sample to be tested.
  • the second reagent may include a dye selected from membrane-specific dyes and mitochondrial-specific dyes, and/or a nucleic acid-specific dye.
  • the membrane-specific dyes can be selected from one or more of DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, Super Fluor 488 and deformed structures based on them.
  • the membrane-specific dye is Alexa Fluor 488.
  • the mitochondrial specific dye can be selected from one or more of Janus Green B, MitoLite Red, Rhodamine 123 and Mitotracker series and their parent.
  • the mitochondrial specific dye is Mitotracker Deep Red or Mitotracker Red.
  • the deformed structure of the dye includes a commercial deformed structure or a non-commercial deformed structure.
  • a commercial deformed structure or a non-commercial deformed structure According to the name and structure of the dye, those skilled in the art can confirm from the prior art the deformation using a known dye as the matrix. Structures (such as commercial deformed structures); at the same time, non-commercial deformed structures can be obtained based on the matrix structure and/or existing deformed structures, and it can be reasonably expected that these deformed structures can achieve a dyeing effect similar to that of the matrix. These deformed structures all fall into the protection scope of the present invention.
  • membrane-specific dye refers to a fluorescent dye capable of specifically staining platelet membranes; similarly, “mitochondrial specific dye” refers to a fluorescent dye capable of specifically staining platelet mitochondria.
  • the number of organelle particles emitted from the reticulocyte has a certain correlation with the number of RET. Since reticulocytes are red blood cells that contain nucleic acid substances, after adding fluorescent dyes, especially nucleic acid-specific dyes, these particles can be specifically dyed.
  • reticulocyte interference to platelets.
  • the above-mentioned second reagent containing membrane or mitochondrial specific dyes can be used to alarm the reticulocytes against this phenomenon.
  • particles related to reticulocytes ie, organelle particles scattered after lysis of reticulocytes
  • the reticulocyte alarm can be carried out for further examination of the subject.
  • the second embodiment of the first aspect of the present invention further includes: a scatter diagram formed according to the forward scattered light intensity information and the fluorescence intensity information in the optical information of the sample to be tested When the number of particles in the preset area exceeds a predetermined threshold, it indicates that there are reticulocytes in the sample to be tested.
  • the mitochondrial dye Mitotracker Deep Red and a conventional hemolytic agent are used to process the blood sample to be tested containing reticulocytes to obtain the sample to be tested, which is then analyzed by blood cells
  • the instrument (Mindray BC6200) was used for measurement.
  • the optical detection device of Mindray BC6200 was modified, and the excitation wavelength of the laser light source was set to 450nm.
  • Figure 6A shows a two-dimensional scatter diagram composed of forward scattered light intensity information and fluorescence intensity information, in which only the ghost area is shown;
  • Figure 6B shows a two-dimensional scatter diagram composed of side scattered light intensity information and fluorescence intensity information Point map.
  • the platelets can be identified in the completely ghosted area through the forward scattered light intensity information and the fluorescence intensity information, while the mature red blood cell area and the reticulocyte area partially overlap. Even so, it can still pass through the preset area. The number of particles indicates the presence of reticulocytes in the test sample. It can be seen from Figure 6B that this method has no effect on the white blood cell count.
  • the white blood cell subpopulations can be distinguished by the information of side scattered light intensity and fluorescence intensity, and monocyte subpopulations (MON), lymphocyte subpopulations (LYM), and neutrophils can be obtained.
  • MON monocyte subpopulations
  • LYM lymphocyte subpopulations
  • neutrophils can be obtained.
  • the second reagent may comprise a nucleic acid specific dye, especially a dye specific to the nucleic acid of reticulocytes.
  • This preferred solution can stain blood samples with nucleic acid dyes, so that not only the information of reticulocytes can be obtained, but also the platelets can be further distinguished from organelle particles scattered after the lysis of reticulocytes.
  • the preferred solution of the present invention includes the ability to further realize the effective measurement of reticulocytes while detecting platelets.
  • steps S131, S132 and S133 of the method are the same as steps S121 and S122 in Fig. 5, wherein the fluorescent dye of the second reagent includes a nucleic acid dye.
  • the forward scattered light intensity information and the fluorescence intensity information can be used to further distinguish the particles related to the reticulocytes, that is, the organelle particles after the lysis of the reticulocytes, that is to say, the test object can be obtained.
  • Optical information of platelets and reticulocytes in the sample can be used to further distinguish the particles related to the reticulocytes, that is, the organelle particles after the lysis of the reticulocytes, that is to say, the test object can be obtained.
  • the particles related to the reticulocytes can be further counted (step S134) to obtain the relative count value of the reticulocytes.
  • the second embodiment according to the first aspect of the present invention can distinguish the to-be-tested sample according to the scattered light intensity information in the optical information of the sample to be tested, especially the forward-scattered light intensity information and the fluorescence intensity information.
  • the platelets and reticulocytes in the sample are used to obtain the optical information of the platelets and the reticulocytes in the sample to be tested. Further, the number of reticulocytes in the sample to be tested can also be estimated based on the optical information of the reticulocytes.
  • nucleic acid fluorescent dyes can also effectively stain leukocyte nuclei, and the use of fluorescent signals can also achieve classification and detection of leukocytes.
  • the nucleic acid-specific dye used in the present invention is not particularly limited.
  • Commercial nucleic acid fluorescent dyes and nucleic acid-specific fluorescent dyes disclosed in some patent applications can be used in the present invention.
  • the commercially available nucleic acid fluorescent dyes include Thermofisher's SYTO series of nucleic acid dyes.
  • the fluorescent dyes disclosed in Chinese patent application CN201010022414.6, the anthocyanin dyes disclosed in CN200910109215.6, the fluorescent dyes disclosed in CN200810216864.1, etc. can all be used in the present invention.
  • the entire contents of the above patent documents are incorporated into this application by reference.
  • the concentration range of the nucleic acid dye varies according to the nature of the dye used, and is not particularly limited, and is usually 0.002 ppm to 2000 ppm.
  • the preferred concentration range is 0.03 ppm to 20 ppm.
  • the second reagent preferably further contains an organic solvent.
  • the organic solvent may be methanol, ethanol, glycerin, etc., but is not limited thereto.
  • the fluorescent dye SYTO9 (Thermofisher company) and a conventional hemolytic agent are used to process the blood sample to be tested containing reticulocytes to obtain the sample to be tested, and then the blood cell analyzer (Mindray BC6200) is used for measurement , The optical detection device of Mindray BC6200 was modified, and the excitation wavelength of the laser light source was set to 520nm. Obtain the forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information of the particles in the sample to be tested to draw a two-dimensional scatter plot of the sample to be tested, as shown in Figs. 8A and 8B.
  • Figure 8A shows a two-dimensional scatter diagram composed of forward scattered light intensity information and fluorescence intensity information, in which only the ghost area is shown;
  • Figure 8B shows a two-dimensional scatter diagram composed of side scattered light intensity information and fluorescence intensity information.
  • Point map It can be seen from Figure 8A that the blood shadow area can be clearly divided into the platelet area, the mature red blood cell area and the reticulocyte area through the forward scattered light intensity information and the fluorescence intensity information, so that the platelets can be accurately counted and the reticulocytes can be counted relatively. . It can be seen from Figure 8B that this method also has no effect on the white blood cell count.
  • the white blood cell subpopulations can be distinguished by the information of the side scattered light intensity and the fluorescence intensity to obtain monocyte subpopulations (MON), lymphocyte subpopulations (LYM), and medium Sex granulocyte subpopulation (NEU), eosinophil subpopulation (EOS).
  • MON monocyte subpopulations
  • LYM lymphocyte subpopulations
  • NOU medium Sex granulocyte subpopulation
  • EOS eosinophil subpopulation
  • the second reagent may include one of membrane or mitochondrial specific dyes and nucleic acid specific dyes to obtain more accurate and precise platelet counts, and at the same time obtain the classification count of white blood cells and reticulocytes. Relative count.
  • platelets can be distinguished from other particles in the sample based on the information of fluorescence and forward scattered light, and the platelet count can be obtained (and in the case of nucleic acid dyes, the network can be obtained).
  • Relative count of erythrocytes and further use the information of fluorescence and side-scattered light to obtain classification information and count of white blood cells.
  • the intensity of fluorescence, forward scattered light and side scattered light can also be used at the same time to obtain a three-dimensional scatter plot of the volume distribution, thereby completing the classification and counting of each particle. Since the three-dimensional scatter plot reflects the characteristics of particles from a multi-dimensional perspective, the discrimination of each particle group is better, and the result is more accurate.
  • the blood detection method further includes counting platelets based on the obtained optical information of the platelets.
  • the blood detection method of the first aspect of the present invention may further eliminate the use of lasers for platelet lysis after performing red blood cell lysis on the blood sample according to the first or second embodiment.
  • the pulse wave of the light signal produces interference in the optical detection device for detection to obtain a more accurate platelet count.
  • FIGS. 11 and 12 are respectively schematic diagrams of the composition structure of a specific example of the optical detection device provided by the present invention.
  • the optical detection device 200 includes an optical subsystem 1, a flow chamber 2 and a detector 3.
  • the optical subsystem 1 includes: a laser 11 (ie, the light source of the present invention), a front light assembly 12 including an optical isolator 121, and a rear light assembly 13 including a straight-blocking diaphragm 131.
  • the laser 11 is configured to emit a laser beam with a wavelength less than 600nm, especially less than 488nm, or is configured to emit violet light, blue light, green light, or yellow light
  • the front light assembly 12 is configured to perform front light treatment on the laser beam, and The laser beam processed by the front light is converged at the straight stop 131 in the second direction, and is converged at the blood cell sample to be measured in the flow cell 2 in the first direction and generates scattered light
  • the rear light assembly 13 is along the laser The propagation direction of the light beam is set after the flow cell 2, and is configured to perform post-light processing on the scattered light and the laser beam converged at the straight stop 131, so that the scattered light after the post-light processing enters the first detector 3 Perform light intensity detection;
  • the first detector 3 of the optical detection device 200 is a forward scattered light detector.
  • the optical detection device 200 may further include a second detector 4.
  • the second detector 4 may be a side scattered light detector, or a medium-angle or high-angle scattered light detector.
  • the optical detection device 200 optionally further includes a fluorescence detector 5.
  • the front light assembly 12 further includes a collimating lens 122, which is arranged between the laser 11 and the optical isolator 121 along the propagation direction (optical axis direction) of the laser beam, and is configured to perform the operation on the laser beam.
  • the collimation process turns the laser beam into a parallel beam.
  • the front light assembly 12 further includes a first light focusing assembly 123 and a second light focusing assembly 124.
  • the first light focusing assembly 123 is configured to perform a first focus on the laser beam so that the The laser beam converges on the blood cell sample under test in the flow cell in the first direction and generates scattered light;
  • the second light converging component 124 is configured to perform a second focus on the laser beam so that the laser beam is in the second direction Converged at the straight aperture 131.
  • the rear light assembly 13 further includes a third converging component 132 and a small aperture diaphragm 133.
  • the third converging component 132 is configured to perform a third focus on the scattered light, so that the scattered light is concentrated on the At the aperture diaphragm, and enter the detector through the aperture of the aperture diaphragm for light intensity detection.
  • optical detection device Specific embodiments of the optical detection device are recorded in the applicant's previous international applications PCT/CN2019/084660 and PCT/CN2019/084509, the complete contents of which are incorporated herein by reference.
  • the second aspect of the present invention provides yet another blood detection method. Referring to FIG. 13, which shows a schematic flow chart of the method, the steps of the method are described.
  • the blood sample is processed with a first reagent and a second reagent to obtain a sample to be tested.
  • the first reagent includes a hemolytic agent that lyses the red blood cells in the blood sample. It is fragments and keeps the cell morphology of leukocytes and platelets in the blood sample substantially intact, and the second reagent includes a fluorescent dye.
  • step S22 the particles in the sample to be tested are allowed to pass through the detection area of the optical detection device one by one, and the light source of the optical detection device is used to irradiate the particles in the sample to be tested to obtain the Optical information of the sample.
  • the light source is configured to emit light with a wavelength of less than 600 nm, especially less than 488 nm, or to emit violet light or blue light or green light or yellow light.
  • step S23 the optical information of the reticulocytes in the sample to be tested is obtained according to the fluorescence intensity information and the scattered light intensity information in the optical information of the sample to be tested.
  • the third aspect of the present invention provides yet another blood testing method.
  • the blood detection method of the third aspect is further described below with reference to FIG. 14.
  • Figure 14 shows a schematic flow chart of this method.
  • step S31 the blood sample to be tested is processed, for example, diluted with a diluent to prepare the first sample to be tested.
  • step S32 the electrical signal of the first sample to be tested is obtained by the impedance detection device. Specifically, the first sample to be tested is allowed to flow in a flow chamber having a hole with electrodes, and the electrical signal generated when the particles in the first sample solution to be tested passes through the hole is detected.
  • step S33 a first detection result of platelets in the first sample to be tested, that is, an impedance method detection result, is obtained according to the electrical signal measured in step S32.
  • step S34 it is determined whether the platelets indicated by the first detection result are abnormal.
  • the first detection result indicates that there is no abnormality in the platelets in the blood sample to be tested.
  • the first detection result indicates that the platelets in the blood sample to be tested are abnormal.
  • a second sample to be tested of the blood sample to be tested is prepared by adding, for example, a diluent or a second sample to be tested is prepared from the first sample to be tested. Furthermore, in steps S36 to S38, the second sample to be tested is treated with a first reagent containing a hemolytic agent to obtain optical information of the second sample to be tested, and at least two light intensities in the optical information The information obtains the second detection result of the second sample to be tested. That is, in steps S36 to S38, the above-mentioned blood detection method provided by the first aspect of the present invention is used to obtain the second detection result of platelets in the second sample to be tested, that is, the optical detection result.
  • the abnormality is that the number of platelets in the blood sample is less than a predetermined threshold, that is, there are low-value platelets or platelet aggregation in the blood sample.
  • step S39 the final test result of the platelets in the blood sample to be tested is obtained according to the first test result and the second test result, Or directly determine the second detection result as the final detection result of platelets in the blood sample to be tested.
  • the invention also provides another method for comparing and correcting the results of platelet measurement by impedance method.
  • this method regardless of whether there is abnormality of platelets in the blood sample to be tested, the platelets in the blood sample to be tested are respectively detected in the impedance method detection channel and the optical detection channel for hemolysis at the same time.
  • the blood sample can be divided into two, one for impedance detection, wherein the impedance detection method is a conventional method, and will not be repeated here; the other can be for optical detection, and the optical detection method is the above-mentioned first aspect of the present invention Blood testing methods.
  • the platelet count result of the impedance method and the platelet count result of the optical method can be obtained at the same time.
  • the platelet detection method of the present invention can obtain an accurate platelet count, under normal circumstances, the platelet counts measured by the two methods should be very close.
  • the result of the optical method is selected to report the platelet count Value, or the platelet count result of the optical method is used to modify the platelet count result of the impedance method; and when the difference between the platelet count result of the impedance method and the platelet count result of the optical method is less than the predetermined threshold, the detection result of the impedance method is accurate , So you can report the result of the impedance method and/or the result of the optical method.
  • a fourth aspect of the present invention provides a blood analysis system, the blood analysis system comprising:
  • a sampling device having a pipette with a pipette nozzle and a driving device for driving the pipette to quantitatively suck blood samples through the pipette nozzle;
  • the sample preparation device has a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the blood sample drawn by the sampling device, and the reagent supply part provides the first reagent to the reaction tank, so that the sampling
  • the blood sample drawn by the device is mixed with the first reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested, wherein the first reagent includes a hemolytic agent, and the hemolytic agent will Lysing the red blood cells in the blood sample into fragments and keeping the cell morphology of the white blood cells and platelets in the blood sample basically intact;
  • the optical detection device includes a light source, a flow chamber and at least two types of detectors.
  • the particles of the sample to be tested can flow in the flow chamber, and the light emitted by the light source irradiates the particles in the flow chamber to produce optical Information, the detector is used to collect the optical information, wherein the light source is configured to emit light with a wavelength of less than 600nm, especially less than 488nm, or emit purple or blue, green or yellow light; and
  • a data processing device that is electrically connected to the optical detection device and includes a processor and a computer-readable storage medium storing a computer program, wherein the data processing device is configured to, when the computer program is executed by the processor, Each step of the method of the first aspect or the second aspect of the present invention detailed above is performed.
  • the aforementioned processor may be a central processing unit, or other general-purpose processors, digital signal processors, application specific integrated circuits, ready-made programmable gate arrays or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components, and the like.
  • the general-purpose processor may be a microprocessor or the processor may also be any conventional processor or the like.
  • the above-mentioned computer-readable storage medium may be volatile memory or non-volatile memory, and may also include both volatile and non-volatile memory.
  • non-volatile memory can be read-only memory, programmable read-only memory, erasable programmable read-only memory, electrically erasable programmable read-only memory, magnetic random access memory, flash memory, magnetic surface Storage, optical disk, or read-only optical disk; magnetic surface storage can be magnetic disk storage or tape storage.
  • Volatile memory may be random access memory, which is used as an external cache.
  • RAM random access memory
  • static random access memory synchronous static random access memory
  • dynamic random access memory synchronous dynamic random access memory
  • synchronous dynamic random access memory double data rate synchronous dynamic random access memory Access memory
  • enhanced synchronous dynamic random access memory synchronous connection dynamic random access memory
  • direct memory bus random access memory direct memory bus random access memory.
  • Fig. 15 shows a specific blood analysis system according to the present invention.
  • the blood analysis system includes a first housing 100, a second housing 200, a sampling device 10, a sample preparation device 30, an optical detection device 50, a data processing device 70, and an output unit 90.
  • the output unit 90 may be a user interface.
  • the optical detection device 50 and the data processing device 70 are arranged inside the second housing 200, and are respectively arranged on both sides of the second housing 200.
  • the sample preparation device 30 is arranged inside the first housing 100, and the output part 90 and the sampling device 10 are arranged on the outer surface of the first housing 100.
  • the sampling device 10 has a sampling needle for collecting a blood sample and transporting the collected blood sample to the sample preparation device 30.
  • the sampling device can collect multiple blood samples, provide them to different chambers of the sample preparation device for different processing, and then perform different tests.
  • the sample preparation device 30 has a reaction tank and a reagent supply part.
  • the reagent supply part stores reagents for reacting with the blood sample (for example, at least stores the aforementioned first reagent and optional second reagent) and supplies corresponding reagents to The reaction tank.
  • the sample preparation device 30 may include at least one reaction cell, wherein the at least one reaction cell may be configured to allow the blood sample from the sampling part to react with the reagent from the reagent supply part to obtain a test solution containing a plurality of platelet particles, so that The platelet particles flow through the flow chamber of the optical detection device one by one.
  • the optical detection device 50 may include: the above-mentioned optical sub-system with a light source, a flow chamber, and at least two detectors.
  • the light source can emit a wavelength of less than 600 nm, especially less than 488 nm, or can emit purple light or blue light or green light or yellow light.
  • the flow chamber allows blood particles such as platelet particles to pass through in a queue.
  • At least two detectors are used to collect optical information of blood particles passing through the flow chamber, especially light intensity information.
  • the at least two detectors include a first detector that detects the forward scattered light intensity of particles flowing in the flow chamber, that is, a forward scattered light detector.
  • the first optical detector is usually arranged on a straight line where the light source and the flow chamber are located, and the light source is arranged on both sides of the flow chamber, respectively.
  • the at least two detectors also include a second detector. The second detector is arranged at a certain angle with the straight line where the light source and the flow chamber are located, so as to detect the side scattered light intensity, the middle angle scattered light intensity or the high angle scattered light intensity of the particles flowing in the flow chamber.
  • the data processing device 70 is configured to detect the blood (for example, platelet) particles flowing through the flow chamber based on the light intensity signals of the at least two kinds of scattered light, and obtain the detection result of the corresponding blood particles.
  • the blood for example, platelet
  • the output unit 90 is configured to output a detection result corresponding to the blood (for example, platelet) particles.
  • the at least two detectors include a forward scattered light detector and a side scattered light detector, and the data processing device is configured to
  • the computer program is executed by the processor, each step of the blood testing method of the first embodiment of the first aspect of the present invention is further executed.
  • the reagent supply part is configured to further provide a second reagent to the reaction cell, so that the blood sample drawn by the sampling device is in contact with the second reagent provided by the reagent supply part.
  • the second reagent includes a fluorescent dye
  • the optical detection device further includes a fluorescence detector
  • the data processing device is configured to When the device is executed, each step of the blood testing method of the second embodiment of the first aspect of the present invention is further executed.
  • the data processing device of the blood analysis system uses fluorescence intensity information and forward scattered light intensity information to further distinguish platelets from reticulocytes or their fragments. Therefore, the blood analysis system can accurately count platelets. The blood analysis system can further obtain the information of the reticulocytes, and then alarm the abnormal condition of the reticulocytes or count the reticulocytes.
  • the data processing device of the blood analysis system can also use the fluorescence intensity, the forward scattered light intensity and the side scattered light intensity to obtain a three-dimensional scatter diagram, so as to better distinguish the platelet particle cluster from other particle clusters and obtain more Accurate platelet count.
  • the optical detection device has a light source 101, a beam shaping component 102, a flow chamber 103, and a forward scattered light detector (ie, a first detector) 104 which are sequentially arranged in a straight line.
  • a dichroic mirror 106 is arranged at an angle of 45° to the straight line. Part of the lateral light emitted by the particles in the flow chamber 103 passes through the dichroic mirror 106, and is arranged at an angle of 45° to the dichroic mirror 106.
  • the fluorescence detector behind the dichroic mirror 106 (that is, the third detection) The other part of the side light is reflected by the dichroic mirror 106, and is arranged at an angle of 45° to the dichroic mirror 106.
  • the side scattered light detector (that is, the second detector) is arranged in front of the dichroic mirror 106. ) 107 capture.
  • a blood analysis system including:
  • a sampling device having a pipette with a pipette nozzle and a driving device for driving the pipette to quantitatively suck blood samples through the pipette nozzle;
  • the sample preparation device has a reaction cell and a reagent supply part, wherein the reaction cell is used to receive the blood sample sucked by the sampling device, and the reagent supply part provides the first reagent and the second reagent to the reaction cell, thereby
  • the blood sample drawn by the sampling device is mixed with the first reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested, wherein the first reagent includes a hemolytic agent, and The hemolytic agent lyses the red blood cells in the blood sample into fragments and keeps the cell morphology of the white blood cells and platelets in the blood sample substantially intact, and the second reagent includes a fluorescent dye;
  • the optical detection device includes a light source, a flow chamber, a scattered light detector, and a fluorescence detector.
  • the particles of the sample to be tested can flow in the flow chamber, and the light emitted by the light source illuminates the particles in the flow chamber
  • the scattered light detector is used to collect scattered light intensity information in the optical information
  • the fluorescence detector is used to collect fluorescence intensity information in the optical information, wherein the light source is configured to emit less than 600nm, especially light less than 488nm or emit blue or violet or green or yellow light; and
  • a data processing device that is electrically connected to the optical detection device and includes a processor and a computer-readable storage medium storing a computer program, wherein the data processing device is configured to, when the computer program is executed by the processor, Each step of the blood testing method of the second aspect of the present invention is executed.
  • the present invention provides a further blood analysis system.
  • the blood analysis system may include an impedance detection device 150 in addition to the above-mentioned components.
  • the impedance detection device 150 includes a flow chamber 151 having a hole 152 with an electrode 153.
  • the impedance detection device 150 detects the DC impedance generated when the particles in the sample to be tested pass through the hole 152, and outputs an electrical signal reflecting the information when the particles pass through the hole.
  • the sampling device 10 is driven by its driving device after the blood sample is drawn and moved to the reaction tank of the sample liquid preparation device 30, and the drawn blood sample is injected into the reaction tank.
  • the conveying device 130 can also convey the sample to be tested after being treated with the diluent in the reaction cell to the impedance detection device 150, that is, to the flow chamber 151.
  • the impedance detection device 150 may also be provided with a sheath fluid tank (not shown) for supplying sheath fluid to the flow chamber 151.
  • the sample liquid to be tested flows under the sheath liquid, the small hole 152 turns the flow of the sample liquid to be tested into a trickle, so that the particles (formed parts) contained in the sample to be tested pass through the small hole 152 one by one. .
  • the electrode 153 is electrically connected to a DC power supply 154, and the DC power supply 154 supplies DC power between the pair of electrodes 153. During the period when the DC power supply 154 provides DC power, the impedance between the pair of electrodes 153 can be detected.
  • the resistance signal representing the change in impedance is amplified by the amplifier 155 and sent to the data processing device 70.
  • the size of the resistance signal corresponds to the volume (size) of the particle. Therefore, the data processing device 70 performs signal processing on the resistance signal to obtain the result of counting platelets in the sample to be tested.
  • the data processing device 70 is configured to implement each step of the blood detection method of the third aspect of the present invention, which will not be repeated here.

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Abstract

一种血液检测方法和相应的血液分析系统，该方法包括：用含有溶血剂的第一试剂处理血液样本以获得待测试样（S11），其中溶血剂将血液样本中的红细胞裂解为碎片；通过光学检测装置（50，200）获得待测试样的光学信息（S12），其中使待测试样中的粒子逐个通过光学检测装置（50，200）的检测区并且利用光学检测装置（50，200）的光源（101）对待测试样中的粒子进行照射，以获取待测试样的光学信息，其中光源（101）配置为发射波长小于488nm的光；和根据光学信息中的至少两种获得血小板的光学信息（S13）和/或网织红细胞的光学信息，其中在裂解血液样本中的红细胞的检测通道中获取血小板和/或网织红细胞的光学信息。

Description

血液检测方法及血液分析系统 技术领域
本发明涉及血液检测，特别涉及血小板的光学检测方法及其血液分析系统。
背景技术
人血液中含有红细胞、白细胞、血小板等各种细胞，其中血小板是直径为2-3微米的无核细胞，正常人的血液中含有15万至35万个/微升血小板。
众所周知，采用血液分析仪对血液样本中的血小板进行测量的方法通常有阻抗法和光学法。
其中阻抗法是基于库尔特原理，使经过稀释的血样中的粒子逐个通过小孔，在小孔的两侧施加恒流源，每一个通过小孔的细胞都引起小孔内液体电阻抗的变化，从而生成电脉冲，然后将检测到的电脉冲绘制成直方图进行分析。正常的血液中，血小板的体积最小，白细胞体积最大，红细胞体积居中。检测到的脉冲强度与经过小孔的细胞体积相关，因而通过体积划分，就能够区分不同细胞种类。然而，在测试有些特殊样本时(如含有体积较大的血小板和体积较小的红细胞的样本)会影响血小板的检测准确性和精确度。这些特殊样本通常来自患有疾病的受试者，因而检测值的偏差会给临床诊断带来不利影响。此外，在阻抗法中，对于低值PLT样本，其PLT直方图与RBC直方图交界处往往没有明显界限，导致算法无法准确切割PLT与RBC直方图，进而不能获取准确的PLT测量结果。
为克服该缺陷，已提出了光学法来测定PLT。光学法基于流式细胞术，其中经稀释和染色的样本在鞘流的挤压下，使样本中的细胞粒子依次通过光学检测区。每个细胞经过激发光源的照射，在光学检测装置中获得代表细胞体积信息的前向散射信号以及被荧光染色剂染色的粒子的荧光信号，以此生成前向散射信号与荧光信号的二维散点图，进而对RBC和PLT进行划分与计数。
流式细胞技术可以迅速测定血液中的细胞，比如美国专利US 6,114,173、US 4,882,284及US 5,891,731中都公开了在非溶血条件下，利用染料对血细胞进行染色以更好地区分出血小板的方法。中国专利申请CN 101173921中公开了一种特异性染色剂用于区分出血小板。
虽然光学法可以获得准确的PLT测量结果，但是需要血液细胞分析仪增加一个专门的检测通道，导致单个样本测量时间延长，测试速度降低。另外，这个专门的检测通道还需要专用的稀释液和染料，因此，设备成本和检测成本均较高，不利于在临床的推广。
因此，仍存在进一步改进血小板的测定方法的需求。
发明内容
针对以上情况，本发明的目的在于提供一种不需单独设置专门的检测通道就能检测血液样本中的血小板的检测方法，该方法利用溶血通道、尤其是常规的白细胞检测通道和特定波长的光源，根据光学信息将血液样本中的血小板从通常被认为是干扰信号的血影粒子中准确地识别出来。
本发明进一步的目的是，根据光学信息尤其是利用荧光信息对具有异常含量的网织红细胞的样本进行报警或识别并计数网织红细胞。
本发明的再一目的是利用本发明检测的血小板数量与常规阻抗法检测的血小板计数结果进行比较和校正。
此外，本发明的目的还在于提供实施上述方法的血液检测系统。
为实现上述目的，本发明第一方面首先提供一种血液检测方法，所述方法包括：
用第一试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息，其中所述光源配置为发射波长小于488nm的光或者发射紫光或蓝光；和
根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
本发明第二方面提供一种血液检测方法，所述方法包括：
用第一试剂和第二试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料；
使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息，其中所述光源配置为发射波长小于488nm的光或者发射紫光或蓝光；和
根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
本发明第三方面提供一种血液检测方法，所述方法包括：
制备含待测血液样本和稀释液的第一待测试样；
使所述第一待测样本液在具有一带电极的孔的流动室中流动并检测所述第一待测样本液中的粒子通过所述孔时产生的电信号；
根据所述电信号求取所述第一待测样本液中的血小板的第一检测结果；
当所述第一检测结果表明所述待测血液样本中的血小板异常时，
制备含所述待测血液样本和稀释液的第二待测样本液或者从所述第一待测样本液制备第二待测样本液；
用第一试剂处理所述第二待测样本液，所述第一试剂包括溶血剂，所述溶血剂至少将所述第二待测样本液中的成熟红细胞完全裂解为碎片并且使所述第二待测样本液中的白细胞和血小板的细胞形态基本保持完整；
使所述第二待测样本液中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述第二待测样本液中的粒子进行照射，以获取所述第二待测样本液的光学信息，其中所述光源配置为发射波长小于488nm的光或者发射紫光或蓝光；以及
根据所述第二待测样本液的光学信息中的至少两种光强度信息获得所述第二待测样本液中的血小板的第二检测结果。
本发明第四方面提供一种血液分析系统，所述血液分析系统包括：
采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
光学检测装置，包括光源、流动室和至少两种检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述检测器用于收集所述光学信息，其中所述光源配置为发射波长小于488nm的光或者发射紫光或蓝光；和
数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
本发明第五方面提供一种血液分析系统，所述血液分析系统包括：
采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂和第二试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料；
光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述散射光检测器用于收集所述光学信息中的散射光强度信息，所述荧光检测器用于收集所述光学信息中的荧光强度信息，其中所述光源配置为发射波长小于488nm的光或者发射紫光或蓝光；和
数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
本发明提供了一种新的检测血小板的方法，该方法不需增加额外的检测通道或使用额外的特异性检测试剂就可获得准确的血小板信息。该方法采用发射波长在488nm以下的光的光源，利用溶血检测通道，检测经溶血处理的血液样本，实现了通过光学检测就能够将血小板完全从血影粒子中区分出来。此外该方法还能够同时获得白细胞的分析结果，并在使用荧光染料的情况下进一步获得网织红细胞的检测信息，从而能够在同一检测通道中同时获得白细胞，甚至网织红细胞的检测信息，从而简化了血液检测、降低了检测成本。
附图说明
图1为根据本发明第一方面的血液检测方法的流程示意图；
图2为采用常规溶血剂处理待测血液样本获得的前向散射光-侧向散射光二维散点图；
图3为根据本发明第一方面的第一实施方式的血液检测方法的流程示意图；
图4为根据本发明第一方面的第一实施方式获得的前向散射光-侧向散射光二维散点图；
图5为根据本发明第一方面的第二实施方式的一个实例的血液检测方法的流程示意图；
图6A和图6B分别为根据本发明第一方面的第二实施方式的一个实例获得的二维散点图；
图7为根据本发明第一方面的第二实施方式的另一个实例的血液检测方法的流程示意图；
图8A和图8B分别为根据本发明第一方面的第二实施方式的另一个实例获得的二维散点图；
图9为根据本发明方法测得的血小板计数值PLT-1与采用单独RET通道测得的血小板计数值PLT-0相关性对比图；
图10根据本发明方法测得的网织红细胞相对计数值RET-1与采用单独RET通道测得的网织红细胞计数值RET-0线性关系对比图；
图11为本发明实施例提供的光学检测装置的组成结构示意图一；
图12为本发明实施例提供的光学检测装置的组成结构示意图二；
图13为根据本发明第二方面的血液检测方法的流程示意图；
图14为根据本发明第三方面的血液检测方法的流程示意图；
图15为本发明实施例提供的血细胞分析系统的组成结构示意图；
图16为本发明实施例提供的血细胞分析系统中的光学检测装置的示意图；
图17为本发明实施例提供的血细胞分析系统中的阻抗检测装置的示意图。
具体实施方式
下面将结合本发明实施方式中的附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明的一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
需要说明的是，在本发明实施例中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者装置不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者是还包括为实施方法或者装置所固有的要素。在没有更多限制的情况 下，由语句“包括一个……”限定的要素，并不排除在包括该要素的方法或者装置中还存在另外的相关要素(例如方法中的步骤或者装置中的单元，这里的单元可以是部分电路、部分处理器、部分程序或软件等等)。
需要说明的是，本发明实施例所涉及的术语“第一\第二\第三”仅仅是区别类似的对象，不代表针对对象的特定排序，可以理解地，“第一\第二\第三”在允许的情况下可以互换特定的顺序或先后次序。应该理解“第一\第二\第三”区分的对象在适当情况下可以互换，以使这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。
如前所述，检测血小板的方法中，常规采用电阻抗法，然而电阻抗法在检测一些特殊血样时，对血小板的检测不够准确。为此，已报道了用光学检测方法结合特定的检测试剂在单独的检测通道中对血小板进行检测。然而这些方法增加了检测设备的成本，也增加了检测费用。
美国专利US 7,344,890 B2中公开了一种采用血影剂(ghosting reagent)处理含有干扰物的血液样品，使红细胞的散射特性发生改变，从而可通过测定样品中细胞的前向散射光的强度和飞行时间，在所得的二维散点图中明显地将血小板与红细胞区分开。该方法通过使正常红细胞失去血红素，从而显著地改变了红细胞的折射率。然而该方法不能有效区分大尺寸血小板和白细胞。而且该方法需要测定飞行时间，并不能仅由光学信息对血小板进行检测。
本发明提出了一种在对血液样本进行溶血处理的前提下，通过光学方法检测血小板的方法，该方法能够利用光学信息使血小板与经溶血处理而裂解的红细胞完全区分开，并且还能同时获得白细胞的光学信息。此外，在采用荧光染料的情况下能进一步获得网织红细胞的光学信息，以便提示在血液样本中存在网织红细胞。
以下对本发明提供的血液检测方法及相应的血液分析系统示例性地详细说明。
根据本发明第一方面的血液检测方法，采用发射紫光或蓝光或绿光或黄光的光源或者说发射波长小于600纳米、尤其是小于488纳米的光的光源进行检测，以便将血液样本中的血小板从血影区中的其他粒子中完全区分开，从能够获得准确的血小板计数。
具体地，参见图1，其中示出了根据本发明第一方面的血液检测方法的流程图。所述血液检测方法包括以下步骤。
步骤S11中，用第一试剂处理血液样本以获得待测试样。该步骤中，第一试剂包括溶血剂，以将所述血液样本中的红细胞裂解为碎片，而血小板可基本保持其细胞形态，优选白细胞也能基本保持其细胞形态。
在步骤S12中，通过光学检测装置获得待测试样中每个粒子的光学信息。具体地，使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息。
在步骤S13中，根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
在本发明实施例中，紫光的波长范围约为370nm～435nm，蓝光的波长范围约为436nm～500nm，绿光的波长范围约为501nm～560nm，黄光的波长范围约为561nm～599nm。在一个实施例中，本发明检测系统的光源允许发射波长约在370nm～435nm范围内、优选在375nm～420nm范围内、更优选在400nm～410nm范围内的光。在另一个实施例中，本发明检测系统的光源允许发射波长约在436nm～500nm范围内、优选在440nm～500nm范围内、更优选在445nm～490nm范围内的光。在又另一实施例中，本发明检测系统的光源允许发射波长约在501nm～560nm范围内、优选在510nm～550nm范围内、更优选在510nm～530nm范围内的光。
在一个实施例中，本发明检测系统的光源可以配置为发射波长约为375nm或405nm或450nm或520nm的光。
更进一步地，根据本发明第一方面的方法在常规的白细胞分类和/或计数通道中实现，对白细胞的分类和/或计数不产生影响。在所获得的散点图(可进一步参考以下详述的实施例)中，白细胞区域显著远离血小板的区域。因而，本发明的方法也不会产生白细胞干扰大尺寸血小板和血小板聚集体的情况。因此，本发明的方法能够同时获得至少三分类(例如单核细胞、淋巴细胞和中性粒细胞)、甚至在利用荧光染料的情况下可获得四分类(例如，淋巴细胞、单核细胞、中性粒细胞和嗜酸性粒 细胞)的白细胞检测结果，并可对网织红细胞报警，或者计数。
进一步地，根据本发明第一方面的血液检测方法中所用的光源可以配置为发射波长约为375nm～480nm范围内的光、尤其是405nm～480nm范围内的光、更优选440nm～480nm范围内的光。
根据本发明第一方面的第一实施方式，第一试剂中可以包含能够使红细胞深度裂解的强溶血剂，将血液样本中的红细胞完全裂解为其光散射特性显著不同于血小板的碎片。所述强溶血剂没有特别限制。举例来说，这样的强溶血剂可为烷基糖苷、三萜皂苷、甾族皂苷等等。
一种具体的强溶血剂可为具有通式I的糖苷类化合物：
R-(CH 2) n-CH 3     (I)
其中，R选自由单糖、去氧单糖和多糖所组成的组，n为5～17的整数。
上述糖苷类化合物能够起到快速溶解红细胞的作用。糖苷类化合物是由糖(或多糖)的半缩醛羟基同烷醇的羟基脱水形成的化合物。本发明的溶血剂中糖苷类化合物可以是单一的化合物，也可以是符合上述通式的两种或更多种糖苷类化合物的混合物。
所述通式I的糖苷类化合物在本发明的溶血剂中的浓度根据所选糖苷的性质、反应时间、反应温度和其他成分的使用量而有所不同，通常用量为0.025g/L～10g/L范围内，优选0.1g/L～5.0g/L。
进一步地，第一试剂可以包括具有通式II的非离子型表面活性剂：
R 1-R 2-(CH 2CH 2O) m-H     (II)
其中，R 1为C8-C23的烷基，R 2为-O-、
Figure PCTCN2019104451-appb-000001
或-COO-，m为10～50的整数；和
可选地，至少一种有机酸或其盐，其中所述有机酸或其盐选自由具有至少一个羧基或磺酸基的有机酸及其碱金属的盐所组成的组中。
通式II的非离子型表面活性剂，在一定程度上能够与细胞膜结合，起到保护白细胞和血小板的细胞膜不受前述糖苷类化合物的影响而保持或基本保持其细胞形态的作用。
在本发明中，通式I和通式II的化合物配合使用，一方面能够获得对红细胞的快速深度裂解的效果，另一方面，为了能够有效检测血小板，起到对血小板细胞膜的保护作用。
根据所选取的通式I和通式II的化合物，二者之间的用量比例也有所不同。但是，通常来说，通式I和通式II的化合物的用量比为1:100～1:3，优选1:25～1:5，更优选1:10～1:5。
根据本发明的优选实施方式，所述第一试剂可进一步包括至少一种有机酸或其盐以使白细胞侧散射光的区分度更好。
本发明的第一试剂还可进一步包括常规的添加剂。这些添加剂可根据需要选择性加入，例如(但不限于)缓冲剂、金属螯合剂、渗透压调节剂、防腐剂等。这些试剂均为本领域常用的试剂，只要不妨碍本发明溶血剂中的上述成分发挥作用即可。
本发明的第一试剂的具体实施例记载在本申请人的在先国际申请PCT/CN2019/084660和PCT/CN2019/084648中，其完整内容通过引用的方式结合于此。
根据第一实施方式的第一试剂与血液样本的混合比例并没有特别限制。举例来说，血液样本与第一试剂的体积混合比可为1:40～1:60。溶血反应在诸如40～60℃的温度下，反应15～100秒，优选反应40～80秒。反应温度和时间可根据具体条件进行调节。
通常来说，较高的反应温度和较长的反应时间，有助于获得对于红细胞程度较深的裂解。
在本发明中，红细胞深度裂解(深度溶血)指红细胞被完全裂解为其光散射特性显著不同于血小板的碎片，而血小板可基本保持其细胞形态，优选白细胞也能基本保持其细胞形态。在散射光信号组成的散点图上，血小板和被深度裂解的红细胞碎片能形成完全区分开的两个粒子群。于此相对地，本文中的红细胞常规裂解指使用常规的溶血剂，溶血剂与血液样本反应后，在散射光信号组成的散点图上，被裂解的红细胞碎片与血小板粒子群可能存在重叠，如图2所示。图2仅示出血影区域，由图2可知，网织红细胞碎片区域与血小板区域相互有一定程度的重叠，难以获得对血小板的准确计数。
在本发明中，强溶血剂尤其是能将可能存在的网织红细胞完全裂解为体积更小的光散射特性显著不同于血小板的碎片。而使用常规的溶血剂处理血细胞，虽然白细胞和血小板大致保持完整细胞形态并且成熟红细胞被完全裂解成碎片，但网织红细胞由于其幼稚性，其细胞部分破碎，有些网织 红细胞碎片的体积可能与血小板类似，因而在散射光信号组成的散点图上，网织红细胞碎片与血小板粒子群可能存在重叠。
因此，在本发明第一方面的第一实施方式中，在使用强溶血剂对红细胞深度裂解的情况下，在通过光学检测得到的至少两种散射光强度信息组成的散点图中能够清晰地区分出红细胞碎片区域和血小板区域，实现对血小板的准确和精确的识别和计数。此外，还能获得至少包括单核细胞、淋巴细胞和中性粒细胞的白细胞亚群。
进一步参考图3，其中示出了该第一实施方式中一种具体的血液检测方法的流程示意图。该具体的血液检测方法中，首先在步骤S111中，用如上所述的含有强溶血剂的第一试剂处理血液样本以获得待测试样。接着在步骤S112中，使待测试样中的粒子逐个通过光学检测装置的流动室，从而获得光学信息。在步骤S113中，根据前向和至少一种其他角度的散射光强度信息，将血小板与其他粒子、尤其是完全裂解的红细胞碎片区分开。此外，在步骤S114中，根据待测试样的光学信息中的前向散射光强度信息和侧向散射光强度信息获得所述待测试样中的白细胞的光学信息，以根据所获得的白细胞光学信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群。
在本发明第一方面的第一实施方式的一个实施例中，采用葵基葡萄糖苷对血液样本进行处理得到待测试样，然后用血液细胞分析仪(迈瑞BC6200)进行测定，其中对迈瑞BC6200的光学检测装置进行了更改，将激光器光源的激发波长设为405nm。获取待测试样中粒子的前向散射光强度信息和侧向散射光强度信息，以绘制所述待测试样的二维散点图，如图4所示。图4仅示出血影区域，从图4可以看出，红细胞碎片区域和血小板区域的区分度较大，可以非常清晰地将血小板从血影区域划分出来进行有效的统计。
在步骤S113中，其他角度的散射光强度信息包括侧向散射光强度信息、中角散射光强度信息和高角散射光强度信息中的至少一种。一个实例中，所述至少两种散射光强度信息包括前向散射光强度信息和侧向散射光强度信息。
前向散射光信号的散射角度可以为约1°～10°。中角散射光信号的散射角度可以为约10°～20°。高角散射光信号的散射角度可以为约20°～70°。侧向散射光信号的散射角度可以为约70°～110°。
参考图5，说明根据本发明第一方面的第二实施方式。图5示出了该第二实施方式中的一种具体的血液检测方法的流程图。
在根据本发明第一方面的第二实施方式中，对用于本发明的第一试剂中的溶血剂没有特别限定，可以是如上所述的强溶血剂，也可以是仅使红细胞常规裂解为碎片的溶血剂。在该第二实施方式中，经第一试剂处理的血液样本还使用含有至少一种荧光染料的第二试剂进行处理。参见图5，在步骤S121中，使用含有溶血剂的第一试剂以及含有荧光染料的第二试剂共同处理血液样本从而获得待测试样。
具体地，在本发明第一方面的第二实施方式中，第一试剂可以包含任意的溶血剂，只要能裂解红细胞即可，对其溶血程度没有特别限制，可以是如上所述的强溶血剂，也可以是常规溶血剂。示例性的常规溶血剂例如季铵盐类阳离子表面活性剂(如十四烷基三甲基氯化铵)，但本发明不限于此。
在本发明第一方面的第二实施方式中，由于对溶血程度没有特别的要求，因此可以将溶血反应的时间设置得更短，例如设置在15至30秒的范围内，特别是20秒左右，以便加快样本的检测速度。
在步骤S122中，同样通过光学检测装置获得待测试样的光学信息。在该步骤中，相应地，除了前述的散射光强度信息外，还进一步获得荧光强度信息。
在步骤S123中，根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息、尤其是前向散射光强度信息获得所述待测试样中的血小板的光学信息，以将血小板从其他粒子中区分出来。
进一步地，在步骤S124中，还可以根据所述待测试样的光学信息中的侧向散射光强度和荧光强度信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群和/或识别幼稚粒细胞。
进一步地，在步骤S125中，还可以根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
进一步地，在步骤S126中，当所述网织红细胞的光学信息满足预设条件时，还可以输出在所述待测试样中存在网织红细胞的提示。
在本发明第一方面的第二实施方式中，第二试剂可以包含选自膜特异性染料和线粒体特异性染料中的一种染料，和/或一种核酸特异性染料。
所述膜特异性染料可以选自DiA、DiD、DiI、DiO、DiR、DiS、FDA、Alexa Fluor 488、Super Fluor488及以它们为母体的变形结构中的一种或多种。优选地，所述膜特异性染料为Alexa Fluor 488。
所述线粒体特异性染料可选自Janus Green B、MitoLite Red、罗丹明123和Mitotracker系列及以它们的母体的一种或多种。优选地，所述线粒体特异性染料为Mitotracker Deep Red或Mitotracker Red。
本发明中，染料的变形结构包括商业化的变形结构或非商业化的变形结构，根据染料的名称、结构等，本领域技术人员能够从现有技术中确认出以已知染料为母体的变形结构(如商业化变形结构)；同时，能够根据母体结构和/或已存在变形结构来得到非商业化的变形结构，并可以合理预期这些变形结构能实现与其母体类似的染色效果。这些变形结构均落入本发明的保护范围之中。
本发明中，“膜特异性染料”是指能够对血小板膜进行特异性染色的荧光染料；类似地，“线粒体特异性染料”是指能够对血小板线粒体进行特异性染色的荧光染料。当血液样本经第一试剂和包含膜特异性染料或线粒体特异性染料的第二试剂处理后，血小板和被裂解的红细胞产生了差异性更为显著的荧光特性，因而可通过检测荧光强度和选自前向散射光强度和侧向散射光强度中的至少一种，特别是通过检测荧光强度和前向散射光强度可更进一步地使血小板区分于被裂解的红细胞碎片，即使采用常规溶血剂对血液样本进行处理。
在本发明中，血液样本中的网织红细胞被溶血剂、尤其是强溶血剂处理后，从网织红细胞(RET)中散出的细胞器颗粒的数目与RET的数量有一定的相关性。由于网织红细胞是含有核酸物质的红细胞，因而加入荧光染料、尤其是核酸特异性染料后，就能够对这些粒子进行特异性能染色。
此外，我们在研究中发现，对于一些存在较大量网织红细胞的血液样本，仅仅用两种散射光信号、尤其是前散射光和侧散射光信号进行血小板检测时，特别是在采用常规溶血剂时，会出现网织红细胞对血小板的干扰。可利用上述含膜或线粒体特异性染料的第二试剂，针对该现象进行网织红细胞报警。具体可在前散射光和荧光信号的二维散点图的预设区域对与网织红细胞相关的粒子(即网织红细胞裂解后散落的细胞器颗粒)进行计数，当计数值超过预定值时，可进行网织红细胞报警，以便进一步对受试者进行检查。
因此，在本发明第一方面的第二实施方式的一种具体实例中进一步包括：当根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息构成的散点图的预设区域中的粒子数超过预定阈值时提示在所述待测试样中存在网织红细胞。
在本发明第一方面的第二实施方式的一个实施例中，采用线粒体染料Mitotracker Deep Red和常规溶血剂对含有网织红细胞的待测血液样本进行处理得到待测试样，然后用血液细胞分析仪(迈瑞BC6200)进行测定，其中对迈瑞BC6200的光学检测装置进行了更改，将激光器光源的激发波长设为450nm。获取待测试样中粒子的前向散射光强度信息、侧向散射光强度信息和荧光强度信息，以绘制所述待测试样的二维散点图，如图6A和图6B。图6A示出由前向散射光强度信息和荧光强度信息组成的二维散点图，其中仅示出血影区域；图6B示出由侧向散射光强度信息和荧光强度信息组成的二维散点图。由图6A可知，通过前向散射光强度信息和荧光强度信息能够将血小板完全血影区域中识别出来，而成熟红细胞区域与网织红细胞区域有部分重叠，即便如此，仍然能够通过预设区域中的粒子数对待测试样中存在网织红细胞进行提示。由图6B可知，该方法对白细胞计数没有影响，可以通过侧向散射光强度和荧光强度信息区分白细胞亚群，获得单核细胞亚群(MON)、淋巴细胞亚群(LYM)、中性粒细胞亚群(NEU)、嗜酸性粒细胞亚群(EOS)。
根据本发明第一方面的第二实施方式的另一种具体实例，第二试剂可包含核酸特异性染料，特别是针对网织红细胞的核酸特异性的染料。该优选方案可对血液样本用核酸染料进行染色，这样不但能获得网织红细胞的信息，还能进一步使血小板区分于网织红细胞溶解后散落的细胞器颗粒。
因此，本发明的该优选方案包括能够进一步实现在检测血小板的同时实现对网织红细胞的有效测定。
参考图7说明该具体实例的血液检测方法。如图7所示，该方法的步骤S131和S132以及S133与前述图5中的步骤S121和S122相同，其中第二试剂的荧光染料包括核酸染料。
进一步地，该方法在步骤S133中，利用前向散射光强度信息和荧光强度信息可以进一步区分出网织红细胞相关的粒子，即网织红细胞溶解后的细胞器颗粒，也就是能够获得所述待测试样中的血小板的光学信息和网织红细胞的光学信息。
进一步地，在步骤S133中区分出血小板和网织红细胞后，可进一步对与网织红细胞相关的粒子(步骤S134)进行计数，以得出网织红细胞的相对计数值。
也就是说，根据本发明第一方面的第二实施方式可以根据所述待测样本的光学信息中的散射光强度信息、尤其是前向散射光强度信息和荧光强度信息，区分所述待测试样中的血小板和网织红细胞以得出所述待测试样中的血小板的光学信息和网织红细胞的光学信息。进一步地，还可以根据所述网织红细胞的光学信息对所述待测试样中的网织红细胞的数量进行估计。
此外，这些核酸荧光染料也能对白细胞核有效染色，进而利用荧光信号也能实现对白细胞的分类检测。
用于本发明的核酸特异性染料没有特别限制。商品化的核酸荧光染料及一些专利申请中已经公开的核酸特异性荧光染料均可用于本发明。其中商品化的核酸荧光染料，可列举的有Thermofisher公司的SYTO系列核酸染料。此外，中国专利申请CN201010022414.6中公开的荧光染料、CN200910109215.6中公开的花青素类染料、CN200810216864.1中公开的荧光染料等，均可用于本发明。以上专利文献的全部内容通过引用并入本申请。
所述核酸染料的浓度范围根据具体采用的染料性质而不同，并没有特别限制，通常在0.002ppm到2000ppm。优选的浓度范围为0.03ppm到20ppm。
所述第二试剂优选还包含有机溶剂。所述有机溶剂可为甲醇、乙醇、甘油等，但不限于此。
在一个具体的实施例中，采用荧光染料SYTO9(Thermofisher公司)和常规溶血剂对含有网织红细胞的待测血液样本进行处理得到待测试样，然后用血液细胞分析仪(迈瑞BC6200)进行测定，其中对迈瑞BC6200的光学检测装置进行了更改，将激光器光源的激发波长设为520nm。获取待测试样中粒子的前向散射光强度信息、侧向散射光强度信息和荧光强度信息，以绘制所述待测试样的二维散点图，如图8A和图8B。图8A示出由前向散射光强度信息和荧光强度信息组成的二维散点图，其中仅示出血影区域；图8B示出由侧向散射光强度信息和荧光强度信息组成的二维散点图。由图8A可知，通过前向散射光强度信息和荧光强度信息能够将血影区域明显分成血小板区域、成熟红细胞区域和网织红细胞区域，因此能够对血小板进行准确计数以及对网织红细胞进行相对计数。由图8B可知，该方法同样对白细胞计数没有影响，可以通过侧向散射光强度和荧光强度信息区分白细胞亚群，获得获得单核细胞亚群(MON)、淋巴细胞亚群(LYM)、中性粒细胞亚群(NEU)、嗜酸性粒细胞亚群(EOS)。
此外，按照上述方法对一组随机血样进行分析获得血影区的血小板计数值PLT-1和网织红细胞相对计数值RET-1，同时用血液细胞分析仪的RET独立通道测得相应的血小板计数值PLT-0和网织红细胞计数值RET-0。将两种方法获得的血小板计数值和网织红细胞计数值进行相关性统计，获得拟合直线，如图9和图10所示，可以看到根据本发明方法测得的血小板计数值PLT-1和网织红细胞计数值PLT-1与采用PET单独通道测得的血小板计数值PLT-0和网织红细胞计数PLT-0之间具有良好的线性关系，因而可以取消单独的网织红细胞通道，直接利用白细胞通道实现对血小板的准确计数以及对网织红细胞的报警和相对计数。
在进一步优选的方案中，第二试剂可包含膜或线粒体特异性染料中的一种以及核酸特异性染料，以获得更准确和精确的血小板计数，并同时获得白细胞的分类计数及网织红细胞的相对计数。
在上述利用含有荧光染料的第二试剂的方法中，可根据荧光和前向散射光的信息使血小板与试样中的其他粒子区分开，获得血小板的计数(以及在使用核酸染料情况下获得网织红细胞的相对计数)，并进一步利用荧光和侧向散射光的信息获得白细胞的分类信息和计数。进一步地，还可同时利用荧光、前向散射光和侧向散射光的强度，获得体积分布的三维散点图，从而完成各粒子的分类 和计数。三维散点图由于从多维角度反映出粒子的特征，因而各粒子群的区分度更好，结果更为准确。
在根据本发明第一方面的血液检测方法中，进一步包括，根据所获得血小板的光学信息对血小板计数。
在本发明第一方面的第三实施方式中，本发明第一方面的血液检测方法可在对血液样本按照前述第一实施方式或第二实施方式进行红细胞裂解后，进一步采用消除了激光器对血小板光信号的脉冲波产生干扰的光学检测装置中进行检测，以获取更为准确的血小板计数。
如图11和12所示，图11和12分别为本发明提供的光学检测装置的一个具体实例的组成结构示意图，光学检测装置200包括光学子系统1、流动室2和检测器3。
光学子系统1包括：激光器11(即本发明的光源)、包括光隔离器121的前光组件12及包括挡直光阑131的后光组件13。其中，激光器11配置为发射波长小于600nm、尤其是小于488nm的激光光束或配置为发射紫光或蓝光或绿光或黄光；前光组件12配置为对所述激光光束进行前光处理，经所述前光处理的激光光束在第二方向上汇聚于挡直光阑131处，在第一方向上汇聚于流动室2的血细胞被测样本处并产生散射光；后光组件13沿所述激光光束的传播方向设置于流动室2之后，配置为对所述散射光及汇聚于挡直光阑131处的激光光束进行后光处理，使得经所述后光处理的散射光进入第一检测器3进行光强检测；光隔离器121配置为隔离所述激光光束经流动室及后光组件所产生的反射光。
光学检测装置200的第一检测器3是前向散射光检测器。光学检测装置200还可包括第二检测器4。第二检测器4可为侧向散射光检测器，也可为中角度或高角度散射光检测器。光学检测装置200可选地还包括荧光检测器5。
在一实施例中，前光组件12还包括准直透镜122，沿所述激光光束的传播方向(光轴方向)设置于激光器11与光隔离器121之间，配置为对所述激光光束进行准直处理，使所述激光光束成为平行光束。
在一实施例中，所述前光组件12还包括第一光汇聚组件123及第二光汇聚组件124，第一光汇聚组件123，配置为对所述激光光束进行第一聚焦，使所述激光光束在第一方向上汇聚于流动室的血细胞被测样本处并产生散射光；第二光汇聚组件124配置为对所述激光光束进行第二聚焦，使所述激光光束在第二方向上汇聚于档直光阑131处。
在一实施例中，后光组件13还包括第三汇聚组件132及小孔光阑133，第三汇聚组件132配置为对所述散射光进行第三聚焦，使所述散射光汇聚于所述小孔光阑处，并经小孔光阑的小孔进入检测器，以进行光强检测。
该光学检测装置的具体实施例记载在本申请人的在先国际申请PCT/CN2019/084660和PCT/CN2019/084509中，其完整内容通过引用的方式结合于此。
本发明第二方面提供又一种血液检测方法。参考图13，其中示出了该方法的流程示意图，说明本方法的步骤。
如图13所示，步骤S21中，用第一试剂和第二试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料。
步骤S22中，使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息。在该方法中，所述光源配置为发射波长小于600nm、尤其是小于488nm的光或者发射紫光或蓝光或绿光或黄光。
最后在步骤S23中，根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
以上详述的本发明第一方面的第二实施方式可全部应用于本发明第二方面的血液检测方法中，在此不再赘述。
本发明的第三方面提供再一种血液检测方法。以下参考图14进一步说明该第三方面的血液检测方法。图14示出了该方法的流程示意图。
步骤S31中，对待测血液样本进行处理，例如用稀释液进行稀释，以制备第一待测试样。
在步骤S32中，通过阻抗检测装置获得所述第一待测试样的电信号。具体地，使第一待测试样在具有一带电极的孔的流动室中流动并检测所述第一待测样本液中的粒子通过所述孔时产生的电信号。
在步骤S33中，根据步骤S32中测得的电信号求取所述第一待测试样中的血小板的第一检测结果，即阻抗法检测结果。
在步骤S34中，判断第一检测结果所示的血小板是否存在异常。当所述第一检测结果表明所述待测血液样本中的血小板无异常时，则报告该第一检测结果。当所述第一检测结果表明所述待测血液样本中的血小板异常时，则进一步进行步骤S35～S39。
在步骤S35中，通过加入例如稀释液制备所述待测血液样本的第二待测试样或者从所述第一待测试样制备第二待测试样。进一步地在步骤S36～S38中，用含有溶血剂的第一试剂处理该第二待测试样，获得第二待测试样的光学信息，并由所述光学信息中的至少两种光强度信息得到该第二待测试样的第二检测结果。即在步骤S36～S38中采用本发明第一方面提供的上述血液检测方法获得所述第二待测试样中的血小板的第二检测结果，即光学法检测结果。
进一步地，所述异常为所述血液样本中的血小板的数量小于预定阈值，即所述血液样本中存在低值血小板或血小板聚集。
进一步地，当第一检测结果表明所述待测血液样本中的血小板异常时，在步骤S39中，根据第一检测结果和第二检测结果求取待测血液样本中的血小板的最终检测结果，或者直接将第二检测结果确定作为待测血液样本中的血小板的最终检测结果。
本发明还提供另一种对阻抗法测定血小板的结果进行比对和校正的方法。在该方法中，无论待测血液样本中是否存在血小板异常，同时在阻抗法检测通道和溶血的光学检测通道中分别检测待测血液样本中的血小板。其中，血液样本可分为两份，一份进行阻抗检测，其中阻抗检测方法为常规方法，在此不再赘述；另一份可进行光学检测，光学检测方法为上述的本发明第一方面的血液检测方法。
在该方法中，可同时获得阻抗法的血小板计数结果和光学法的血小板计数结果。
由于本发明的血小板检测方法能够获得准确的血小板计数，因此正常情况下，两种方法测得的血小板计数应该是非常接近的。当阻抗法的血小板计数结果和光学法的血小板计数结果之差大于等于预定阈值时，则可判定该样本的血小板含量异常，阻抗法的结果不准确，因而选择光学法的结果来报告血小板的计数值，或者采用光学法的血小板计数结果修正阻抗法的血小板计数结果；而当阻抗法的血小板计数结果和光学法的血小板计数结果之差小于预定阈值时，则说明阻抗法的检测结果是准确的，因此可报告阻抗法的结果和/或光学法的结果。
本发明第四方面提供一种血液分析系统，所述血液分析系统包括：
采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
光学检测装置，包括光源、流动室和至少两种检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述检测器用于收集所述光学信息，其中所述光源配置为发射波长小于600nm、尤其是小于488nm的光或者发射紫光或蓝光或绿光或黄光；和
数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以上详述的本发明第一方面或第二方面的方法的各个步骤。
上述处理器可以是中央处理单元，还可以是其他通用处理器、数字信号处理器、专用集成电路、现成可编程门阵列或者其他可编程逻辑器件、分立门或者晶体管逻辑器件、分立硬件组件等。通用 处理器可以是微处理器或者该处理器也可以是任何常规的处理器等。
上述计算机可读存储介质可以是易失性存储器或非易失性存储器，也可包括易失性和非易失性存储器两者。其中，非易失性存储器可以是只读存储器、可编程只读存储器、可擦除可编程只读存储器、电可擦除可编程只读存储器、磁性随机存取存储器、快闪存储器、磁表面存储器、光盘、或只读光盘；磁表面存储器可以是磁盘存储器或磁带存储器。易失性存储器可以是随机存取存储器，其用作外部高速缓存。通过示例性但不是限制性说明，许多形式的RAM可用，例如静态随机存取存储器、同步静态随机存取存储器、动态随机存取存储器、同步动态随机存取存储器、双倍数据速率同步动态随机存取存储器、增强型同步动态随机存取存储器、同步连接动态随机存取存储器、直接内存总线随机存取存储器。本发明实施方式描述的存储器旨在包括这些和任意其它适合类型的存储器。
图15示出了根据本发明的一种具体的血液分析系统。该血液分析系统包括第一机壳100、第二机壳200、采样装置10、样本制备装置30、光学检测装置50、数据处理装置70及输出部90。在实际应用中，输出部90可以为用户界面。本实施方式中，光学检测装置50及数据处理装置70设置在第二机壳200的内部，分别设置在第二机壳200两侧。样本制备装置30设置在第一机壳100的内部，输出部90和采样装置10设置在第一机壳100的外表面。
采样装置10具有采样针，用于采集血液样本并将采集的血液样本输送至样本制备装置30。根据不同的实施方式，采样装置可采集多份血液样本，提供给样本制备装置的不同腔室进行不同的处理，并随后进行不同的检测。
样本制备装置30具有反应池和试剂供应部，试剂供应部贮存用于与血液样本反应的试剂(例如至少储存有前述第一试剂和可选的第二试剂)并根据需要将相应的试剂供应到所述反应池。
样本制备装置30可包括至少一个反应池，其中至少一个反应池可配置为使得来自采样部的所述血液样本和来自试剂供应部的试剂进行反应，得到包含多个血小板粒子的待测试液，使得所述血小板粒子逐一流经光学检测装置的流动室。
所述光学检测装置50可包括：上述具有光源的光学子系统、流动室和至少两个检测器。所述光源可发射波长小于600nm、尤其是小于488nm，或者可发送紫光或蓝光或绿光或黄光。所述流动室，供诸如血小板粒子的血液粒子排队通过。至少两个检测器用于收集经过流动室的血液粒子的光学信息，尤其是光强度信息。
在本发明的装置中，所述至少两个检测器包括检测在流动室中流动的粒子的前向散射光强度的第一检测器，即，前向散射光检测器。该第一光学检测器通常布置在光源和流动室所在的直线上，和光源分别布置在流动室的两侧。所述至少两个检测器还包括第二检测器。所述第二检测器与所述光源和流动室所在的直线呈一定角度布置，以检测在流动室中流动的粒子的侧向散射光强度、中角度散射光强度或高角度散射光强度。
数据处理装置70配置为根据至少两种散射光的光强信号，检测出流经所述流动室的血液(例如，血小板)粒子，得到对应血液粒子的检测结果。
输出部90配置为输出对应所述血液(例如血小板)粒子的检测结果。
根据本发明前述的方法，在第一试剂包含强溶血剂的情况下，所述至少两种检测器包括前向散射光检测器和侧向散射光检测器，且所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行上述本发明第一方面的第一实施方式的血液检测方法的各个步骤。
在另一实施例中，所述试剂供应部配置为进一步将第二试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第二试剂在所述反应池中混合，以制备待测试样，所述第二试剂包括荧光染料，所述光学检测装置还包括荧光检测器，所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行上述本发明第一方面的第二实施方式的血液检测方法的各个步骤。
根据本发明的上述方法，该血液分析系统的数据处理装置利用荧光强度信息和前向散射光强度信息，可进一步使血小板与网织红细胞或其碎片相区分。从而该血液分析系统可对血小板准确计数。该血液分析系统还可进一步获得网织红细胞的信息，并进而对网织红细胞存在异常的情况进行报警或对网织红细胞进行计数。
该血液分析系统的数据处理装置还可同时使用荧光强度、前向散射光强度和侧向散射光强度获得三维散点图，从而更好地将血小板粒子群与其他粒子群区分开，获得更为准确的血小板计数。
进一步参见图16，其中示出了上述血液分析系统中的光学检测装置的一个具体实例。如图16所示，该光学检测装置具有依次布置在一条直线上的光源101、光束整形组件102、流动室103和前向散射光检测器(即第一检测器)104。在流动室103的一侧，与所述直线成45°角布置有二向色镜106。通过流动室103中的粒子发出的侧向光，一部分透过二向色镜106，被与二向色镜106成45°角布置在二向色镜106后面的荧光检测器(即第三检测器)105捕获；另一部分侧向光被二向色镜106反射，被与二向色镜106成45°角布置在二向色镜106前面的侧向散射光检测器(即第二检测器)107捕获。
根据本发明的第五方面，提供一种血液分析系统，包括：
采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂和第二试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料；
光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述散射光检测器用于收集所述光学信息中的散射光强度信息，所述荧光检测器用于收集所述光学信息中的荧光强度信息，其中所述光源配置为发射波长小于600nm、尤其是小于488nm的光或者发射蓝光或紫光或绿光或黄光；和
数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行上述本发明第二方面的血液检测方法的各个步骤。
本发明提供进一步的血液分析系统。如图17所示，所述血液分析系统除了包含上述部件之外，还可以包括阻抗检测装置150。阻抗检测装置150包括具有一带电极153的孔152的流动室151。阻抗检测装置150检测待测试样中的粒子通过所述孔152时产生的直流阻抗，并输出反映粒子通过孔时的信息的电信号。
具体地，采样装置10在吸取血液样本之后由其驱动装置驱动并移动至样本液制备装置30的反应池，将所吸取的血液样本注入到该反应池中。输送装置130还可以将在反应池中经稀释液处理后的待测试样输送到阻抗检测装置150中，即输送到流动室151中。阻抗检测装置150还可以设有未图示的鞘液舱，用于给流动室151提供鞘液。在流动室152中，待测样本液在鞘液的包裹下流过，小孔152使待测样本液流变为细流，使待测试样中所含粒子(有形成份)逐一通过小孔152。电极153与直流电源154电连接，直流电源154向一对电极153之间提供直流电。在直流电源154提供直流电期间，可以检出一对电极153间的阻抗。表示阻抗变化的电阻信号被放大器155放大后输送到数据处理装置70。电阻信号的大小与粒子的体积(大小)相对应，因此通过数据处理装置70对电阻信号进行信号处理可以获得待测试样中的血小板的计数结果。
在该血液分析系统中，数据处理装置70配置用于实施上述本发明第三方面的血液检测方法的各个步骤，在此不再赘述。
以上提及的特征，只要在本发明的范围内是有意义的并且不会相互矛盾，均可以任意相互组合。针对本发明的血液分析方法所说明的优点和特征以相应的方式适用于本发明相应的血液分析系统。
以上所述仅为本发明的优选实施方式，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。

Claims (36)

  1. 一种血液检测方法，包括：
    用第一试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
    使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息，其中所述光源配置为发射波长小于488nm的光；和
    根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
  2. 一种血液检测方法，包括：
    用第一试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
    使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息，其中所述光源配置为发射紫光或蓝光；和
    根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
  3. 根据权利要求1或2所述的血液检测方法，其中所述光源配置为发射波长约为375nm～480nm范围内的光、尤其是405nm～480nm范围内的光、更优选440nm～480nm范围内的光。
  4. 根据权利要求3所述的血液检测方法，其中所述光源配置为发射波长约为375nm或405nm或450nm的光。
  5. 根据权利要求1至4中任一项所述的血液检测方法，其中所述溶血剂为强溶血剂，所述强溶血剂将所述血液样本中的红细胞完全裂解为其光散射特性显著不同于血小板的碎片。
  6. 根据权利要求5所述的血液检测方法，其中所述至少两种光强度信息包括前向散射光强度信息，并且包括侧向散射光强度信息、中角散射光强度信息和高角散射光强度信息中的至少一种，以将所述待测试样中的血小板与其他粒子、尤其是完全裂解的红细胞碎片区分开。
  7. 根据权利要求6所述的血液检测方法，其中所述至少两种光强度信息包括前向散射光强度信息和侧向散射光强度信息，以将所述待测试样中的血小板与其他粒子、尤其是完全裂解的红细胞碎片区分开；和/或
    所述方法进一步包括：
    根据所述待测试样的光学信息中的前向散射光强度信息和侧向散射光强度信息获得所述待测试样中的白细胞的光学信息，以根据所获得的白细胞光学信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群。
  8. 根据权利要求1至5中任一项所述的血液检测方法，所述方法进一步包括：
    在进行光学检测之前用第二试剂处理所述血液样本，所述第二试剂包括荧光染料，相应地，所述待测试样的光学信息进一步包括荧光强度信息；
    其中根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息、尤其是前向散射光强度信息获得所述待测试样中的血小板的光学信息，以识别所述待测试样中的血小板。
  9. 根据权利要求8所述的血液检测方法，其中所述方法进一步包括：
    根据所述待测试样的光学信息中的侧向散射光强度和荧光强度信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群和/或识别幼稚粒细胞。
  10. 根据权利要求8或9所述的血液检测方法，其中所述方法进一步包括：
    根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
  11. 根据权利要求10所述的血液检测方法，其中所述方法进一步包括：
    当所述网织红细胞的光学信息满足预设条件时，输出在所述待测试样中存在网织红细胞的提示。
  12. 根据权利要求11所述的血液检测方法，其中所述荧光染料包括选自膜特异性染料和线粒体特异性染料中的一种；
    所述方法进一步包括：当根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息构成的散点图的预设区域中的粒子数超过预定阈值时提示在所述待测试样中存在网织红细胞。
  13. 根据权利要求11或12所述的血液检测方法，其中所述荧光染料包括核酸特异性染料，优选地，所述核酸特异性染料为对网织红细胞的核酸特异性染料；
    其中根据所述待测试样的光学信息中的荧光强度信息和前向散射光强度信息获得所述待测试样中的血小板的光学信息和网织红细胞的光学信息。
  14. 根据权利要求13所述的血液检测方法，其中所述方法进一步包括：根据所述网织红细胞的光学信息，估计所述待测试样中的网织红细胞的数量。
  15. 根据权利要求1～14中任一项所述的血液检测方法，其中所述方法进一步包括：根据所获得血小板的光学信息对血小板计数。
  16. 一种血液检测方法，包括：
    用第一试剂和第二试剂处理血液样本以获得待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料；
    使所述待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述待测试样中的粒子进行照射，以获取所述待测试样的光学信息，其中所述光源配置为发射波长小于488nm的光，或者发射蓝光或紫光；和
    根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
  17. 根据权利要求16所述的血液检测方法，其中所述方法进一步包括：
    根据所述待测试样的光学信息中的侧向散射光强度和荧光强度信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群和/或识别幼稚粒细胞。
  18. 根据权利要求16或17所述的血液检测方法，其中所述方法进一步包括：
    当所述网织红细胞的光学信息满足预设条件时，输出在所述待测试样中存在网织红细胞的提示。
  19. 根据权利要求16至18中任一项所述的血液检测方法，其中所述荧光染料包括选自膜特异性染料和线粒体特异性染料中的一种；
    所述方法进一步包括：当根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息构成的散点图的预设区域中的粒子数超过预定阈值时提示在所述待测试样中存在网织红细胞。
  20. 根据权利要求13至19中任一项所述的血液检测方法，其中所述荧光染料包括核酸特异性染料，优选地，所述核酸特异性染料为对网织红细胞的核酸特异性染料；
    其中根据所述待测试样的光学信息中的荧光强度信息和前向散射光强度信息，区分所述待测试样中的血小板和网织红细胞以获得所述待测试样中的网织红细胞的光学信息。
  21. 根据权利要求20所述的血液检测方法，其中所述方法进一步包括：根据网织红细胞的光学信息，估计所述待测试样中的网织红细胞的数量。
  22. 一种血液检测方法，其中所述方法包括：
    制备含待测血液样本和稀释液的第一待测试样；
    使所述第一待测试样在具有一带电极的孔的流动室中流动并检测所述第一待测试样中的粒子通过所述孔时产生的电信号；
    根据所述电信号求取所述第一待测试样中的血小板的第一检测结果；
    当所述第一检测结果表明所述待测血液样本中的血小板异常时，
    制备含所述待测血液样本和稀释液的第二待测试样或者从所述第一待测试样制备所述第二待测试样；
    用第一试剂处理所述第二待测试样，所述第一试剂包括溶血剂，所述溶血剂将所述第二待测样本液中的红细胞裂解为碎片并且使所述第二待测试样中的白细胞和血小板的细胞形态基本保持完整；
    使经溶血处理的第二待测试样中的粒子逐个通过光学检测装置的检测区并且利用所述光学检测装置的光源对所述第二待测试样中的粒子进行照射，以获取所述第二待测试样的光学信息，其中所述光源配置为发射波长小于488nm的光，或者发射蓝光或紫光；和
    根据所述第二待测试样的光学信息中的至少两种光强度信息获得所述第二待测试样中的血小板的第二检测结果。
  23. 根据权利要求22所述的血液检测方法，其中所述异常为所述第一待测试样中的血小板的数量小于预定阈值。
  24. 根据权利要求22或23所述的血液检测方法，其中所述方法进一步包括：当所述第一检测结果表明所述待测血液样本中的血小板异常时，根据所述第一检测结果和所述第二检测结果求取所述待测血液样本中的血小板的最终检测结果，或者将所述第二检测结果确定为所述待测血液样本中的血小板的最终检测结果。
  25. 一种血液分析系统，包括：
    采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
    样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整；
    光学检测装置，包括光源、流动室和至少两种检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述检测器用于收集所述光学信息，其中所述光源配置为发射波长小于488nm的光，或者发射紫光或蓝光；和
    数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述待测试样的光学信息中的至少两种光强度信息获得所述待测试样中的血小板的光学信息。
  26. 根据权利要求25所述的血液分析系统，其中所述光源配置为发射波长约为375nm～480nm范围内的光、尤其是405nm～480nm范围内的光、更优选440nm～480nm范围内的光。
  27. 根据权利要求26所述的血液分析系统，其中所述光源配置为发射波长约为375nm或405nm或450nm的光。
  28. 根据权利要求25至27中任一项所述的血液分析系统，其中所述溶血剂为强溶血剂，所述强溶血剂将所述血液样本中的红细胞完全裂解为其光散射特性显著不同于血小板碎片，所述至少两种检测器包括前向散射光检测器和侧向散射光检测器，并且所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行以下步骤：
    根据所述待测试样的光学信息中的前向散射光强度信息和侧向散射光强度信息，将所述待测试样中的血小板与其他粒子、尤其是完全裂解的红细胞碎片区分开；和/或
    根据所述待测试样的光学信息中的前向散射光强度信息和侧向散射光强度信息将所述待测试样中的白细胞至少区分为单核细胞、淋巴细胞和中性粒细胞亚群。
  29. 根据权利要求25至27中任一项所述的血液分析系统，其中所述试剂供应部配置为进一步将第二试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第二试剂在所述反应池中混合，以制备待测试样，所述第二试剂包括荧光染料，所述光学检测装置还包括荧光检测器，所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行以下步骤：
    根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息、尤其是前向散射光强度信息识别所述待测试样中的血小板，和/或
    根据所述待测试样的光学信息中的侧向散射光强度和荧光强度信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群和/或识别幼稚粒细胞，和/或
    据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织 红细胞的光学信息。
  30. 根据权利要求29所述的血液分析系统，其中所述荧光染料包括选自膜特异性染料和线粒体特异性染料中的一种，所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行以下步骤：
    当根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息构成的散点图的预设区域中的粒子数超过预定阈值时提示在所述待测试样中存在网织红细胞。
  31. 根据权利要求29或30所述的血液分析系统，其中所述荧光染料包括核酸特异性染料，优选地，所述核酸特异性染料为对网织红细胞的核酸特异性染料，所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行以下步骤：
    根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息，以获得所述待测试样中的血小板的光学信息和网织红细胞的光学信息；以及
    可选地根据所述网织红细胞的光学信息估计所述待测试样中的网织红细胞的数量。
  32. 根据权利要求25至31中任一项所述的血液分析系统，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，进一步执行以下步骤：根据所获得血小板的光学信息对血小板计数。
  33. 一种血液分析系统，包括：
    采样装置，具有带吸移管嘴的吸移管并且具有驱动装置，该驱动装置用于驱动所述吸移管通过所述吸移管嘴定量吸取血液样本；
    样本制备装置，具有反应池和试剂供应部，其中，所述反应池用于接收采样装置所吸取的血液样本，所述试剂供应部将第一试剂和第二试剂提供给所述反应池，从而由所述采样装置所吸取的血液样本与由所述试剂供应部提供的第一试剂在所述反应池中混合，以制备成待测试样，其中，所述第一试剂包括溶血剂，所述溶血剂将所述血液样本中的红细胞裂解为碎片并且使所述血液样本中的白细胞和血小板的细胞形态基本保持完整，所述第二试剂包括荧光染料；
    光学检测装置，包括光源、流动室、散射光检测器和荧光检测器，所述待测试样的粒子可在所述流动室内流动，所述光源所发射的光照射所述流动室中的粒子以产生光学信息，所述散射光检测器用于收集所述光学信息中的散射光强度信息，所述荧光检测器用于收集所述光学信息中的荧光强度信息，其中所述光源配置为发射波长小于488nm的光或者发射蓝光或紫光；和
    数据处理装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的计算机可读存储介质，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述待测试样的光学信息中的荧光强度信息和散射光强度信息获得所述待测试样中的网织红细胞的光学信息。
  34. 根据权利要求33所述的血液检测方法，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：
    根据所述待测试样的光学信息中的侧向散射光强度和荧光强度信息区分白细胞亚群以获得白细胞的至少包括单核细胞、淋巴细胞和中性粒细胞的亚群和/或识别幼稚粒细胞。
  35. 根据权利要求33或34所述的血液检测方法，其中所述荧光染料包括选自膜特异性染料和线粒体特异性染料中的一种，所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：
    当根据所述待测试样的光学信息中的前向散射光强度信息和荧光强度信息构成的散点图的预设区域中的粒子数超过预定阈值时提示在所述待测试样中存在网织红细胞。
  36. 根据权利要求33至35中任一项所述的血液检测方法，其中所述荧光染料包括核酸特异性染料，优选地，所述核酸特异性染料为对网织红细胞的核酸特异性染料，其中所述数据处理装置配置为当所述计算机程序被所述处理器执行时，执行以下步骤：
    根据所述待测试样的光学信息中的荧光强度信息和前向散射光强度信息，区分所述待测试样中的血小板和网织红细胞以获得所述待测试样中的网织红细胞的光学信息；以及
    可选地根据所述网织红细胞的光学信息估计所述待测试样中的网织红细胞的数量。
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