WO2019206300A1 - 一种血液检测方法及血液分析系统 - Google Patents

一种血液检测方法及血液分析系统 Download PDF

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Publication number
WO2019206300A1
WO2019206300A1 PCT/CN2019/084660 CN2019084660W WO2019206300A1 WO 2019206300 A1 WO2019206300 A1 WO 2019206300A1 CN 2019084660 W CN2019084660 W CN 2019084660W WO 2019206300 A1 WO2019206300 A1 WO 2019206300A1
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blood
light
laser beam
scattered light
sample
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PCT/CN2019/084660
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English (en)
French (fr)
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陈庚文
张子千
叶燚
李朝阳
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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 EP19792205.7A priority Critical patent/EP3789764A4/en
Priority to CN201980008302.4A priority patent/CN111602052B/zh
Publication of WO2019206300A1 publication Critical patent/WO2019206300A1/zh
Priority to US17/081,195 priority patent/US20210102935A1/en

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Electro-optical investigation, e.g. flow cytometers 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
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1429—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2015/018—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present invention relates to blood testing, and more particularly to an optical detection method for platelets and a blood analysis system therefor.
  • Human blood contains various cells such as red blood cells, white blood cells, and platelets.
  • the platelets are non-nuclear cells with a diameter of 2-3 microns, and normal human blood contains 150,000 to 350,000 microliters of platelets.
  • the method is to pass a sample containing blood cells from a small hole having two electrodes.
  • blood cells such as platelets
  • the resistance changes, thereby generating a resistance pulse, and then the detected pulse is drawn 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 medium.
  • the detected pulse intensity is related to the cell volume passing through the well, so that by dividing the volume, it is possible to distinguish between different cell types.
  • platelet detection accuracy and accuracy are affected. These special samples are usually from subjects with disease, so deviations in the measured values can adversely affect clinical diagnosis.
  • Flow cytometry can rapidly determine cells in the blood.
  • U.S. Patent Nos. 6,114,173, 4,882,284, and 5,891,731 the use of dyes to stain blood cells for better differentiation of platelets under non-hemolytic conditions is disclosed.
  • broken red blood cells, lipids, and the like sometimes appear in the blood, and their size is similar to that of platelets, and is also stained, and thus becomes an impurity that affects platelet measurement.
  • the effects of these impurities will be greater.
  • a further object of the present invention is to further detect white blood cells in the blood based on optical information, and to further alarm reticulocytes or detect reticulocytes in the above detection method.
  • Another object of the present invention is to provide a blood test system embodying the above method.
  • the present invention first provides a first 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 comprising a hemolytic agent that cleaves red blood cells in the blood sample into fragments whose light scattering properties are significantly different from those of platelets;
  • Optical information of the platelets is obtained based on at least two of the optical information.
  • the at least two optical information are forward scattered light intensity and side scattered light intensity to distinguish platelets from lysed red blood cell debris.
  • the method further comprises obtaining optical information of the white blood cells based on optical information of the intensity of the forward scattered light and the intensity of the side scattered light, preferably distinguishing the subset of white blood cells according to the obtained optical information of the white blood cells to obtain at least the white blood cells.
  • the hemolytic agent is at least one selected from the group consisting of an alkyl glycoside, a triterpenoid saponin, and a steroidal saponin.
  • the alkyl glycoside is selected from the group consisting of glycoside compounds of the formula I:
  • R is selected from the group consisting of monosaccharides, deoxymonosaccharides, and polysaccharides, and n is an integer of 5-17.
  • the monosaccharide may be selected from the group consisting of five carbon sugars, methyl five carbon sugars, and six carbon sugars, wherein five carbon sugars such as arabinose, xylose, ribose, lyxose, etc.; methyl five carbon sugars such as sucrose, buckthorn Sugar, chicken sugar, etc.; six carbon sugars such as glucose, mannose, fructose, galactose, sorbose; said deoxymonosaccharides such as deoxyribose, deoxyglucose, etc.; said polysaccharides such as maltose, sucrose and the like.
  • n is preferably an integer of 6 to 14, and more preferably an integer of 7 to 11.
  • the first reagent further comprises:
  • Nonionic surfactants of the general formula II are nonionic surfactants of the general formula II:
  • R 1 is a C8-C23 alkyl group, and R 2 is -O-, Or -COO-,m is an integer from 10 to 50;
  • At least one organic acid or a salt thereof wherein the organic acid or a salt thereof is selected from the group consisting of salts of an organic acid having at least one carboxyl group or a sulfonic acid group and an alkali metal thereof.
  • R 1 is a C8-C18 linear alkyl group.
  • the linear alkyl group of C8-C18 may specifically be an octyl group, a sunflower group, a lauryl group, a tetradecyl group, a hexadecyl group or a stearyl group. More preferably, R 1 is a C12-C16 linear alkyl group, and specifically may be lauryl, tetradecyl or hexadecyl. R 2 is preferably -O-. m is preferably from 15 to 30.
  • the first reagent preferably contains 0.025 g/L to 10 g/L, preferably 0.1 g/L to 5.0 g/L of the glycoside compound of the formula I, and 0.03 to 1.5 g/L, preferably 0.05 to 1.0 g/L of a nonionic surfactant of the formula II.
  • the first agent may further comprise one or more selected from the group consisting of a buffer, a metal chelating agent, an osmotic pressure adjusting agent, and a preservative.
  • the volume mixing ratio of the blood sample to the first reagent may be 1:40 to 1:60.
  • the blood sample is treated with the first reagent at a temperature such as 40 to 60 ° C for 15 to 100 seconds, preferably for 40 to 80 seconds.
  • the red blood cells in the sample are deeply cleaved after being treated with the first reagent, and the platelets still maintain their cell morphology, so that the scattered light properties of the obtained red blood cell fragments are significantly different from the scattered light properties of the platelets.
  • a scatter plot obtained from the intensity of forward scattered light and side scattered light enables accurate optical information of the platelets to be obtained, thereby enabling accurate counting of platelets under hemolysis conditions.
  • the method further comprises treating the blood sample with a second reagent comprising a fluorescent dye.
  • the particles in the sample to be tested are passed through the detection area of the optical detection system one by one, and the optical information of the sample to be tested further includes fluorescence information, and is distinguished according to the side scattered light intensity and the fluorescence intensity information.
  • the leukocyte subset obtains at least a subset of leukocytes including monocytes, lymphocytes and neutrophils and/or recognizes immature granulocytes.
  • the fluorescent dye comprises a first fluorescent dye selected from the group consisting of a membrane-specific dye and a mitochondria-specific dye.
  • the membrane-specific dye is selected from the group consisting of DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, Super Fluor 488, and a deformed structure in which they are the parent, and the mitochondria specificity
  • the dye is selected from the group consisting of Janus Green B, MitoLite Red, Rhodamine 123 and Mitotracker series and a deformed structure of their parent.
  • the mitochondria-specific dye is Rhodamine 123, Mitotracker Deep Red or Mitotracker Red.
  • the Mitotracker series of dyes in the present invention may include Mitotracker Green, Mitotracker Deep Red, and Mitotracker Red.
  • platelets are identified based on fluorescence intensity and forward scattered light intensity information.
  • the reticulated red blood cells are alarmed when the number of particles in the predetermined region of the scattergram composed of the forward scattered light intensity and the fluorescence intensity exceeds a predetermined threshold.
  • the fluorescent dye comprises a second fluorescent dye selected from one of nucleic acid-specific dyes.
  • the nucleic acid-specific dye is a dye specific for the nucleic acid of reticulocytes.
  • the method further includes identifying reticulocytes based on fluorescence intensity and scattered light intensity information.
  • the method further comprises performing a reticulocyte count based on the fluorescence intensity and the forward scattered light intensity information.
  • the fluorescent dye comprises a first fluorescent dye selected from one of a membrane-specific dye and a mitochondria-specific dye, and a first one selected from the group consisting of nucleic acid-specific dyes. Two fluorescent dyes.
  • the method further comprises: distinguishing between platelets and reticulocytes based on fluorescence intensity and side scattered light intensity information.
  • the blood detecting method of the present invention described above is capable of counting platelets based on optical information of the obtained platelets.
  • the method of the present invention further detects in an optical detection system capable of eliminating interference of reflected light with a laser, wherein the optical detection system comprises: an optical subsystem, a flow chamber, a first Detector;
  • the optical subsystem includes: a laser, a front light assembly, and a rear light assembly, wherein the front light assembly includes an optical isolator;
  • the laser configured to emit a laser beam
  • the front light assembly is configured to perform front light processing on the laser beam, and the laser beam processed by the front light is concentrated in a first direction at a blood cell sample to be measured in the flow chamber to generate scattered light;
  • the rear light assembly is disposed after the flow chamber along a propagation direction of the laser beam, and is configured to perform post-light processing on the scattered light such that the scattered light processed by the back light enters the first A detector performs light intensity detection;
  • the optical isolator is configured to isolate reflected light from the laser; the reflected light is generated by the laser beam passing through the flow chamber.
  • the optical isolator is composed of a beam splitting prism and a polarization state conversion component which are connected to each other by adhesion;
  • the beam splitting prism is configured to reflect an S-polarized component of the incident laser beam and transmit a P-polarized component of the incident laser beam;
  • the polarization state conversion component is configured to change a polarization state of the P polarization component transmitted through the beam splitting prism, change the P polarization component from linearly polarized light to circularly polarized light, and change the circularly polarized light reflection
  • the latter polarization state is S-polarized light and is reflected by the beam splitting prism.
  • the optical isolator is composed of an analyzer and a polarization conversion component that are connected to each other by adhesion;
  • the analyzer is configured to allow only a P-polarized component of the laser beam to pass;
  • the polarization state conversion component is configured to change a polarization state of the P-polarized component of the analyzer, to change the P-polarization component from linearly polarized light to circularly polarized light, and to change the circularly polarized light
  • the reflected light has a polarization state of S-polarized light and is isolated by the analyzer.
  • the front light assembly further includes a collimating lens; the collimating lens is disposed between the laser and the optical isolator along a propagation direction of the laser beam, and is configured to The laser beam is collimated to make the laser beam a parallel beam.
  • the backlight assembly further includes a straight stop; the front light assembly is further configured to perform a front light treatment on the laser beam such that the laser beam processed by the front light is in a Converging in the two directions directly at the straight stop.
  • the front light assembly further includes a first light concentrating component and a second light concentrating component; the first light concentrating component is configured to perform first focusing on the laser beam to enable the laser The light beam converges in the first direction at the blood cell sample to be measured and generates scattered light; the second light concentrating component is configured to perform a second focus on the laser beam such that the laser beam is at The two directions converge at a straight stop included in the rear light assembly.
  • the backlight assembly further includes a third convergence assembly and a small aperture stop; the third convergence assembly configured to perform a third focus on the scattered light to converge the scattered light
  • the aperture is at the aperture and enters the first detector through the aperture of the aperture.
  • the optical detection system further includes a second detector and a fluorescence detector
  • the second detector is configured to perform light intensity detection on scattered light within an angle range set by the angle of propagation of the laser beam (eg, 60° to 120°);
  • the fluorescence detector is configured to perform fluorescence detection on fluorescence generated by the blood cell sample to be tested.
  • the second direction is perpendicular to a flow direction of the blood cell sample to be tested
  • the first direction is the same as the flow direction of the blood cell sample to be tested.
  • the optical isolator has an optical isolation of not less than 30 db.
  • the light collecting angle of the straight blocking light is 1 to 10 degrees.
  • the wavelength of the laser beam is 630 nm to 640 nm.
  • the laser beam is P-line polarized light.
  • the above optical detecting system can well isolate the reflected light generated when the laser beam propagates in the optical path, so that the laser can stably output the laser beam, thereby preventing the reflected light from entering the small pulse of the power peak generated by the laser, and avoiding the interference.
  • the small pulse and the small pulse formed by the platelet particles are confused with each other, which further improves the detection precision of the platelet by the method of the present invention.
  • the invention also provides a second blood testing method, the method comprising:
  • Optical information of the platelets is obtained from at least two of the optical information.
  • the hemolytic agent cleaves red blood cells in the blood sample into fragments whose light scattering properties are significantly different from those of platelets.
  • the first reagent comprises at least one hemolytic agent selected from the group consisting of an alkyl glycoside, a triterpenoid saponin, and a steroidal saponin.
  • the alkyl glycoside is selected from the group consisting of glycoside compounds of the formula I:
  • R is selected from the group consisting of monosaccharides, deoxymonosaccharides, and polysaccharides, and n is an integer of 5-17.
  • the monosaccharide may be selected from the group consisting of five carbon sugars, methyl five carbon sugars, and six carbon sugars, wherein five carbon sugars such as arabinose, xylose, ribose, lyxose, etc.; methyl five carbon sugars such as sucrose, buckthorn Sugar, chicken sugar, etc.; six carbon sugars such as glucose, mannose, fructose, galactose, sorbose; said deoxymonosaccharides such as deoxyribose, deoxyglucose, etc.; said polysaccharides such as maltose, sucrose and the like.
  • n is preferably an integer of 6 to 14, and more preferably an integer of 7 to 11.
  • the first reagent further comprises a nonionic surfactant of the general formula II:
  • R 1 is a C8-C23 alkyl group, and R 2 is -O-, Or -COO-,m is an integer from 10 to 50;
  • At least one organic acid or a salt thereof wherein the organic acid or a salt thereof is selected from the group consisting of salts of an organic acid having at least one carboxyl group or a sulfonic acid group and an alkali metal thereof.
  • R 1 is a C8-C18 linear alkyl group.
  • the linear alkyl group of C8-C18 may specifically be an octyl group, a sunflower group, a lauryl group, a tetradecyl group, a hexadecyl group or a stearyl group. More preferably, R 1 is a C12-C16 linear alkyl group, and specifically may be lauryl, tetradecyl or hexadecyl. R 2 is preferably -O-. m is preferably from 15 to 30.
  • the first reagent preferably contains 0.025 g/L to 10 g/L, preferably 0.1 g/L to 5.0 g/L of the glycoside compound of the formula I, and 0.03 to 1.5 g/L, preferably 0.05 to 1.0 g/L of a nonionic surfactant of the formula II.
  • the first agent may further comprise one or more selected from the group consisting of a buffer, a metal chelating agent, an osmotic pressure adjusting agent, and a preservative.
  • the volume mixing ratio of the blood sample to the first reagent may be 1:40 to 1:60.
  • the blood sample is treated with the first reagent at a temperature such as 40 to 60 ° C for 15 to 100 seconds, preferably for 40 to 80 seconds.
  • the blood detecting method according to the first embodiment wherein the at least two types of optical information are forward scattered light intensity and side scattered light intensity.
  • the red blood cells in the sample are deeply cleaved after being treated with the first reagent, and the platelets still maintain their cell morphology, so that the scattered light properties of the obtained red blood cell fragments are significantly different from the scattered light properties of the platelets.
  • a scatter plot obtained from the intensity of forward scattered light and side scattered light enables accurate optical information of the platelets to be obtained, thereby enabling accurate counting of platelets under hemolysis conditions.
  • the method further comprises: after treating the blood sample with the first reagent, further processing with a second reagent to obtain a sample to be tested.
  • the second reagent comprises one selected from the group consisting of a membrane-specific dye and a mitochondria-specific dye.
  • the film-specific dye is selected from the group consisting of DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, and Super Fluor 488.
  • the mitochondria-specific dye is selected from the group consisting of Janus Green B, MitoLite Red, Rhodamine 123 and Mitotracker.
  • the mitochondria-specific dye is Rhodamine 123, Mitotracker Deep Red or Mitotracker Red.
  • the Mitotracker series of dyes in the present invention may include Mitotracker Green, Mitotracker Deep Red, and Mitotracker Red.
  • the second agent of the present invention further comprises a deformed structure having the above dye as a parent.
  • the platelets and the lysed red blood cells produce a more pronounced fluorescent characteristic and thus can be detected
  • the fluorescence intensity and at least one selected from the group consisting of forward scattered light intensity and side scattered light intensity, in particular by detecting fluorescence intensity and forward scattered light intensity, can further distinguish platelets from lysed red blood cell fragments.
  • platelets can be identified based on fluorescence intensity and side scattered light intensity information; white blood cell subpopulations can be distinguished according to side scattered light intensity and fluorescence intensity information to obtain at least mononuclear cells, lymphocytes, and neutrals of white blood cells.
  • the second reagent comprises a nucleic acid-specific dye or a dye selected from the group consisting of a membrane-specific dye and a mitochondria-specific dye, and a nucleic acid specific Sex dyes.
  • the organelle scattered after red blood cell lysis is dyed by a nucleic acid-specific dye to further distinguish the fragments of the reticulocytes having the nucleus from the platelets, and at the same time obtain accurate optical information of the reticulocytes, thereby utilizing the first
  • the method of the embodiment can simultaneously obtain optical information of platelets, white blood cells and reticulocytes in one optical detection.
  • the blood detecting method further comprises: distinguishing between platelets and reticulocytes based on fluorescence intensity and scattered light intensity information. More preferably, platelets and reticulocytes are distinguished based on fluorescence intensity and forward scattered light intensity information, and reticulocytes can be counted.
  • the entire contents of the first embodiment in the second blood detecting method are equally applicable to the first blood detecting method of the present invention. Similarly, all of the first blood test methods described above are also applicable to the first embodiment of the second blood test method.
  • the blood detecting method comprises: treating the blood sample with a first reagent and performing treatment with a second reagent to obtain a sample to be tested, wherein the first reagent includes a hemolytic agent.
  • the hemolytic agent is not particularly limited, and a conventional erythrocyte hemolytic agent in the art can be used in this embodiment.
  • the second reagent of this embodiment may be one selected from the above-described membrane-specific dyes and the above-described mitochondria-specific dyes, as in the first embodiment described above.
  • the platelets and the lysed red blood cells produce a more distinct fluorescent property, thus
  • the platelets can be distinguished from the lysed red blood cell fragments by detecting the fluorescence intensity and at least one selected from the group consisting of forward scattered light intensity and side scattered light intensity, particularly by detecting fluorescence intensity and forward scattered light intensity. Due to the use of membrane-specific dyes or mitochondria-specific dyes, in this embodiment, red blood cells can be clearly distinguished from platelets by two-dimensional scattergrams only by lysis of conventional hemolytic agents, without having to red blood cells as in the first embodiment. Perform deep cracking.
  • the leukocyte subset can be distinguished according to the fluorescence intensity and the side scattered light intensity information to obtain a subset of leukocytes including at least monocytes, lymphocytes and neutrophils and/or to recognize immature granulocytes;
  • the fluorescence intensity and the forward scattered light intensity information completely distinguish the platelets; and/or alarm the reticulocytes when the number of particles in the predetermined region of the scattergram based on the forward scattered light intensity and the fluorescence intensity exceeds a predetermined threshold.
  • the second reagent may further comprise a fluorescent dye selected from the group consisting of nucleic acid-specific dyes. And likewise, it is thereby possible to further distinguish between platelets and reticulocytes based on fluorescence intensity and forward scattered light intensity information, preferably further counting reticulocytes.
  • the blood detecting method of the present invention described above can obtain the optical information by detection of an optical detecting system, and the optical detecting system includes:
  • optical subsystem optical subsystem, flow chamber, first detector
  • the optical subsystem includes: a laser, a front light assembly including an optical isolator, and a backlight assembly including a direct aperture stop; wherein
  • the laser configured to emit a laser beam
  • the front light assembly is configured to perform front light processing on the laser beam, and the laser beam processed by the front light is concentrated in the second direction in the direct beam stop, and is concentrated in the first direction
  • the blood cells of the flow cell are measured at the sample and generate scattered light
  • the rear light assembly is disposed after the flow direction of the laser beam, and is configured to perform post-light processing on the scattered light and the laser beam concentrated at the straight stop, so that the light is processed
  • the scattered light of the post-light treatment enters the first detector for light intensity detection;
  • the optical isolator is configured to isolate reflected light generated by the laser beam through the flow chamber and the rear light assembly.
  • the optical isolator is composed of a beam splitting prism and a polarization state conversion component which are connected to each other by adhesion;
  • the beam splitting prism is configured to reflect an S-polarized component of the incident laser beam and transmit a P-polarized component of the incident laser beam;
  • the polarization state conversion component is configured to change a polarization state of the P polarization component transmitted through the beam splitting prism, change the P polarization component from linearly polarized light to circularly polarized light, and change the circularly polarized light reflection
  • the latter polarization state is S-polarized light and is reflected by the beam splitting prism.
  • the optical isolator is composed of an analyzer and a polarization conversion component which are connected to each other by bonding;
  • the analyzer is configured to allow only a P-polarized component of the laser beam to pass;
  • the polarization state conversion component is configured to change a polarization state of the P-polarized component of the analyzer, to change the P-polarization component from linearly polarized light to circularly polarized light, and to change the circularly polarized light
  • the reflected light has a polarization state of S-polarized light and is isolated by the analyzer.
  • the reflectance of the first incident surface of the dichroic prism is not more than 0.5%.
  • the optical isolator is composed of a band pass filter and a frequency doubling crystal which are connected to each other by bonding;
  • the band pass filter is configured to pass the laser beam having a wavelength of ⁇ ;
  • the frequency doubling crystal is configured to multiply a laser beam passing through the band pass filter, and multiply the reflected light of the multiplied laser beam by the band pass filter The pieces are filtered out.
  • the front light assembly further includes a collimating lens
  • the collimating lens is disposed between the laser and the optical isolator along a propagation direction of the laser beam, and is configured to perform collimation processing on the laser beam to make the laser beam into a parallel beam.
  • the front light assembly further includes a first light concentrating component and a second light concentrating component
  • the first light concentrating component is configured to perform a first focusing on the laser beam, so that the laser beam converges in a first direction at a blood cell sample to be measured in the flow chamber and generates scattered light;
  • the second light concentrating assembly is configured to perform a second focusing on the laser beam such that the laser beam converges in the second direction in the direct beam stop.
  • the backlight assembly further includes a third convergence component and a small aperture aperture
  • the third converging component is configured to perform a third focusing on the scattered light, concentrating the scattered light at the aperture stop, and entering the first through the aperture of the aperture aperture Detector.
  • the optical detection system further includes a second detector and a fluorescence detector
  • the second detector is configured to perform light intensity detection on scattered light within an angle range set by the angle of propagation of the laser beam (eg, 60° to 120°);
  • the fluorescence detector is configured to perform fluorescence detection on fluorescence generated by the blood cell sample to be tested.
  • the second direction is perpendicular to a flow direction of the blood cell sample to be tested
  • the first direction is the same as the flow direction of the blood cell sample to be tested.
  • the optical isolator has an optical isolation of not less than 30 db.
  • the light collecting angle of the straight blocking light is 1 to 10 degrees.
  • the wavelength of the laser beam is 630 nm to 640 nm.
  • the laser beam is P-line polarized light.
  • the above optical detecting system can well isolate the reflected light generated when the laser beam propagates in the optical path, so that the laser can stably output the laser beam, thereby preventing the reflected light from entering the small pulse of the power peak generated by the laser, and avoiding the interference.
  • the small pulse and the small pulse formed by the platelet particles are confused with each other, which further improves the detection precision of the platelet by the method of the present invention.
  • the present invention also provides a blood analysis system comprising:
  • a sampling portion configured to acquire a blood sample, and deliver the blood sample to the reaction portion
  • a reagent supply unit for storing at least a first reagent and supplying the reaction portion as needed
  • reaction portion comprising a mixing chamber for mixing the blood sample with a first reagent to form a sample to be tested, wherein the first reagent comprises a hemolytic agent, and the hemolytic agent lyses red blood cells in the blood sample;
  • An optical detection system comprising a flow chamber and at least a first detector for conveying the sample to be tested to the optical system by the mixing chamber and passing particles in the sample to be tested one by one through the flow chamber Upon reaching the detection zone, the first detector detects particles in the test sample to obtain optical information in the sample to be tested;
  • a data processing module operatively coupled to the optical system and including a processor and a non-transitory computer readable storage medium storing a computer program, wherein when the computer program is executed by the processor, performing the following Step: Obtain optical information of the platelets based on at least two of the optical information.
  • the hemolytic agent cleaves red blood cells in the blood sample into pieces whose light scattering properties are significantly different from those of platelets, and wherein the processing module, when the computer program When executed by the processor, the following steps are further performed:
  • the white blood cells are classified into at least a subset including monocytes, lymphocytes, and neutrophils according to the intensity of forward scattered light and the intensity of side scattered light.
  • the mixing chamber is for mixing the blood sample with a second reagent to form a sample to be tested, wherein the second reagent comprises a fluorescent dye selected from the group consisting of a membrane-specific dye and a mitochondria-specific dye;
  • the optical detection system includes a second detector, the second detector being a fluorescence detector to further obtain a fluorescent signal when particles in the sample to be tested pass through the detection zone one by one;
  • leukocytes comprising at least a subset of monocytes, lymphocytes and neutrophils and/or identifying immature granulocytes;
  • the reticulated red blood cells are alarmed when the number of particles in the predetermined region of the scattergram formed based on the forward scattered light intensity and the fluorescence intensity information exceeds a predetermined threshold.
  • the second reagent further comprises a fluorescent dye selected from the group consisting of nucleic acid-specific dyes, and when the computer program is executed by the processor, further performing the following Step: Distinguish between platelets and reticulocytes based on fluorescence intensity and forward scattered light intensity information, preferably further counting reticulocytes.
  • optical detection system further comprises: an optical subsystem
  • the optical subsystem includes: a laser, a front light assembly, and a rear light assembly, wherein the front light assembly includes an optical isolator;
  • the laser configured to emit a laser beam
  • the front light assembly is configured to perform front light processing on the laser beam, and the laser beam processed by the front light is concentrated in a first direction at a blood cell sample to be measured in the flow chamber to generate scattered light;
  • the rear light assembly is disposed after the flow chamber along a propagation direction of the laser beam, and is configured to perform post-light processing on the scattered light such that the scattered light processed by the back light enters the first A detector performs light intensity detection;
  • the optical isolator is configured to isolate reflected light from the laser; the reflected light is generated by at least the laser beam passing through the flow chamber.
  • optical detection system of the present invention is applicable to the optical detection system of the blood analysis system of the present invention.
  • the invention provides a novel blood detecting method, which performs hemolysis treatment on a blood sample, and can accurately detect platelets by optical detection, and the method can simultaneously obtain the analysis result of the white blood cell and use the nucleic acid specificity.
  • the detection information of reticulocytes is further obtained, thereby realizing the use of optical information to distinguish platelets under hemolysis conditions for the first time, and simultaneously obtaining detection information of white blood cells and even reticulocytes in a single channel, thereby simplifying blood detection, Reduced inspection costs.
  • FIG. 1 is a schematic structural diagram 1 of an optical detection system according to an embodiment of the present invention.
  • FIG. 2 is a second schematic structural diagram of an optical detection system according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of the principle of an optical isolator according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of a first incident surface of an optical isolator according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of a light collecting principle of a second cylindrical mirror according to an embodiment of the present invention.
  • Figure 6 is a front elevational view of a straight aperture stop according to an embodiment of the present invention.
  • FIG. 7 is a schematic structural diagram of a blood cell analyzer according to an embodiment of the present invention.
  • FIG. 8 is a schematic structural diagram 3 of an optical detection system according to an embodiment of the present invention.
  • FIG. 9 is a schematic structural diagram 4 of an optical detection system according to an embodiment of the present invention.
  • Figure 10 is a scatter plot of platelet (A) and white blood cells (B) collected by flow cytometry on the deep hemolyzed sample obtained in Example 1;
  • Figure 11 is a comparison of PLT exhibited by the deeply hemolyzed sample (A) of Example 2 and the sample (B) after hemolysis by a conventional hemolytic agent;
  • Figure 12 is a light intensity scatter plot (A) of fluorescence-forward scattered light detected by platelets and reticulocytes in a deep hemolysis state in Example 3, and light intensity scatter of leukocyte fluorescence-side scattered light.
  • Figure (B)
  • Figure 13 is a light intensity scatter plot (A) of fluorescence-forward scattered light detected by platelets and reticulocytes in a deep hemolysis state in Example 4, and light intensity scatter of leukocyte fluorescence-side scattered light.
  • Figure (B)
  • Figure 14 is a light intensity scatter plot (A) of fluorescence-forward scattered light detected by adding a nucleic acid dye to a platelet and reticulocyte in a deep hemolysis state, and fluorescence of a leukocyte-side scattered light.
  • Strength scatter plot (B)
  • Figure 15 is a light intensity scatter plot (A) of fluorescence-forward scattered light detected by adding a membrane dye to a platelet and reticulocyte in a deep hemolysis state, and fluorescence of a leukocyte-side scattered light.
  • Strength scatter plot (B) is a light intensity scatter plot of fluorescence-forward scattered light detected by adding a membrane dye to a platelet and reticulocyte in a deep hemolysis state, and fluorescence of a leukocyte-side scattered light.
  • Figure 16 is a light intensity scatter plot of fluorescence-side scattered light detected by adding mitochondrial dye to platelets and reticulocytes in a deep hemolysis state in Example 7;
  • Figure 17 is a light intensity scatter plot (A) of fluorescence-forward scattering light detected by adding a membrane dye and a nucleic acid dye to platelets and reticulocytes in a deep hemolysis state, and fluorescence-side scattering of leukocytes in Example 8.
  • Light intensity scatter plot (B) of fluorescence-forward scattering light detected by adding a membrane dye and a nucleic acid dye to platelets and reticulocytes in a deep hemolysis state, and fluorescence-side scattering of leukocytes in Example 8.
  • Figure 18 is a diagram showing the correlation between test platelet (A) and reticulocyte (B) in Example 9;
  • Figure 19 is a graph showing the staining and discrimination effect of the dye Alexa Fluor 488 on (A) platelets and (B) leukocytes in the hemolysis state of Example 10.
  • Figure 20 is a graph showing the effect of dye DiD on the staining and discrimination of (A) platelets and (B) leukocytes in the hemolysis state of Example 11.
  • Figure 21 is a graph showing the staining and discrimination effect of dye rhodamine 123 on (A) platelets and (B) leukocytes in the hemolysis state of Example 12.
  • Figure 22 is a graph showing the staining and discrimination effect of the dye Mitotracker Deep Red on (A) platelets and (B) leukocytes in the hemolysis state of Example 13.
  • Figure 23 is a graph showing the staining and discrimination effect of the dye Mitotracker Red on (A) platelets and (B) leukocytes in the hemolysis state of Example 14.
  • Figure 24 is a correlation between the staining method of Example 15 and the test values on the Mindray 6800 instrument.
  • the terms "including”, “comprising”, or any other variations thereof are intended to encompass non-exclusive inclusions, such that a method or apparatus comprising a plurality of elements includes not only the Elements, but also other elements not explicitly listed, or elements that are inherent to the implementation of the method or device.
  • an element defined by the phrase “comprising a " does not exclude the presence of additional related elements in the method or device including the element (eg, a step in the method or a unit in the device)
  • the unit here may be part of a circuit, part of a processor, part of a program or software, etc.).
  • first ⁇ second ⁇ third according to the embodiment of the present invention is only a similar object, and does not represent a specific ordering for an object. It can be understood that “first ⁇ second ⁇ The three” may be interchanged in a specific order or order. It is to be understood that the "first ⁇ second ⁇ third” distinguished objects may be interchanged as appropriate to enable the embodiments of the invention described herein to be carried out in a sequence other than those illustrated or described herein.
  • the electrical impedance method is conventionally used, whereas the electrical impedance method is inaccurate in detecting platelets when detecting some special blood samples.
  • platelets are detected in separate detection channels using optical detection methods in conjunction with specific detection reagents. These methods are performed in separate detection channels without hemolysis.
  • erythrocyte hemolytic agents can also damage other cell membranes while destroying the erythrocyte membrane.
  • red blood cell debris produced after hemolysis is generally considered to cause greater interference with the detection of platelets. Therefore, most of the currently known methods for optically detecting platelets use a method that does not hemolyze.
  • US Patent No. 7,344,890 B2 discloses the use of a ghosting reagent to treat a blood sample containing an interfering substance, thereby changing the scattering characteristics of the red blood cells, thereby determining the intensity and flight of the forward scattered light of the cells in the sample.
  • platelets are clearly distinguished from red blood cells in the resulting two-dimensional scattergram.
  • This method significantly changes the refractive index of red blood cells by causing normal red blood cells to lose heme.
  • this method does not effectively distinguish between large-sized platelets and white blood cells.
  • this method requires measurement of flight time, and it is not possible to detect platelets only by optical information.
  • the present inventors have developed a method for optically detecting platelets under the premise of hemolysis treatment of a blood sample, which is capable of distinguishing platelets from red blood cells lysed by hemolysis treatment using optical information, and simultaneously The optical information of the white blood cells is obtained, and the optical information of the reticulocytes is further obtained with the nucleic acid-specific dye.
  • a blood sample is first treated with a first reagent including a hemolytic agent to obtain a sample to be tested, and then the sample is detected by an optical detecting device, and the platelet is used with at least two kinds of optical information.
  • the other particles in the sample are distinguished to obtain optical information of the platelets. It has been verified that the platelet count obtained by the method of the present invention is highly consistent with the results obtained by other methods of blood analyzers.
  • the method of the present invention does not affect the counting of white blood cells in a conventional white blood cell counting channel.
  • the leukocyte region is significantly distant from the platelet region.
  • the method of the present invention also does not produce a situation in which white blood cells interfere with large-sized platelets and platelet aggregates. Therefore, the method of the present invention is capable of simultaneously obtaining at least three classifications, and even in the case of using a fluorescent dye, four-class white blood cell detection results can be obtained, and reticulocytes can be alarmed or counted.
  • the first reagent contains a hemolytic agent capable of deeply lysing red blood cells.
  • the hemolytic agent is not particularly limited, and the red blood cell deep hemolytic agent of the present invention can be used.
  • such hemolytic agents may be alkyl glycosides, triterpenoid saponins, steroidal saponins, and the like.
  • a specific hemolytic agent can be a glycoside compound of the formula I:
  • R is selected from the group consisting of monosaccharides, deoxymonosaccharides, and polysaccharides, and n is an integer of 5-17.
  • glycoside compound can function to rapidly dissolve red blood cells.
  • a glycoside compound is a compound formed by dehydration of a hemiacetal hydroxyl group of a sugar (or polysaccharide) with a hydroxyl group of an alkanol.
  • 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 conforming to the above formula.
  • the monosaccharide is not particularly limited, and a commonly used one may be selected from the group consisting of five carbon sugars, methyl five carbon sugars, and six carbon sugars, but is not limited thereto.
  • Five carbon sugars such as arabinose, xylose, ribose, lyxose, and the like.
  • Methyl pentasaccharide such as saccharide, rhamnose, chicken sugar and the like.
  • Six carbon sugars such as glucose, mannose, fructose, galactose, sorbose.
  • the deoxymonosaccharide is also not particularly limited, such as, but not limited to, deoxyribose, deoxyglucose, and the like.
  • the polysaccharide is, for example, maltose, sucrose or the like, but is not limited thereto.
  • n is preferably an integer of 6 to 14, and more preferably an integer of 7 to 11.
  • the glycoside compound of the formula I may specifically be an octyl glucoside, a decyl glucoside, a sunflower glucoside, a dodecyl maltoside, a tetradecyl maltoside, a dodecyl glucoside, preferably a octyl group.
  • the concentration of the glycoside compound of the formula I in the hemolytic agent of the present invention varies depending on the nature of the selected glycoside, the reaction time, the reaction temperature, and the amount of other components used, and is usually used in an amount of from 0.025 g/L to 10 g. In the range of /L, it is preferably 0.1 g/L to 5.0 g/L.
  • R 1 is a C8-C23 alkyl group, and R 2 is -O-, Or -COO-,m is an integer from 10 to 50;
  • At least one organic acid or a salt thereof wherein the organic acid or a salt thereof is selected from the group consisting of salts of an organic acid having at least one carboxyl group or a sulfonic acid group and an alkali metal thereof.
  • the nonionic surfactant of the general formula II can bind to the cell membrane to a certain extent, and functions to protect the cell membrane of leukocytes and platelets from the aforementioned glycosides to maintain or substantially maintain their cell morphology.
  • R 1 is a C8-C18 linear alkyl group.
  • the linear alkyl group of C8-C18 may specifically be an octyl group, a sunflower group, a lauryl group, a tetradecyl group, a hexadecyl group or a stearyl group. More preferably, R 1 is a C12-C16 linear alkyl group, and specifically may be lauryl, tetradecyl or hexadecyl.
  • R 2 is preferably -O-. m is from 10 to 50, preferably from 15 to 30.
  • nonionic surfactant of the formula II may be cetyl alcohol polyoxyethylene (15) ether, dodecanol polyoxyethylene (21) ether, cetyl alcohol polyoxyethylene (23) Ether, cetyl alcohol polyoxyethylene (25) ether, cetyl alcohol polyoxyethylene (30) ether, but is not limited thereto.
  • the concentration of the nonionic surfactant of the formula II is not particularly limited, but may be 0.03 to 1.5 g/L, preferably 0.05 to 1.0 g/L.
  • the nonionic surfactant may be used as a single substance or as a mixture of two or more.
  • concentration in the hemolytic agent is also different depending on the kind of the nonionic surfactant to be used. In general, the longer the alkyl chain and the more the number of repeating units of the polyoxyethylene moiety, the lower the concentration of the nonionic surfactant.
  • the compound of the formula I and the formula II can be used in combination to obtain the effect of rapid deep lysis of red blood cells on the one hand, and the protective effect on the platelet cell membrane in order to effectively detect platelets.
  • the ratio between the two will also vary.
  • the compound of the formula I and the formula II is used in an amount of from 1:100 to 1:3, preferably from 1:25 to 1:5, more preferably from 1:10 to 1:5.
  • the first reagent may further comprise at least one organic acid or a salt thereof to make the degree of discrimination of leukocyte side scattered light better.
  • the organic acid is preferably a monohydric, dihydric or tribasic carboxylic acid which is unsubstituted or substituted by a hydroxy or amino group; a C1-6 alkyl sulfonic acid which is unsubstituted or substituted by a hydroxy or amino group. a C6-10 aryl C1-6 alkyl acid; a C6-10 aryl di(C1-6 alkyl acid); and a C6-10 aryl sulfonic acid group.
  • organic acid and its salt may be formic acid, acetic acid, benzoic acid, citric acid (3-hydroxy-1,3,5-pentatritic acid), malic acid (2-hydroxysuccinic acid), benzene Formic acid, benzenesulfonic acid, ⁇ -naphthalenesulfonic acid, taurine, and the like, and alkali metal salts thereof, such as sodium salts and potassium salts, are not limited thereto.
  • the concentration of the organic or organic acid salt in the hemolytic agent is from 0.05 g/L to 2 g/L, preferably from 0.1 g/L to 0.5 g/L.
  • the first agent of the present invention may further comprise a conventional additive.
  • additives may be optionally added as needed, such as, but not limited to, buffers, metal chelators, osmotic pressure adjusting agents, preservatives, and the like. These agents are all commonly used in the art as long as they do not interfere with the action of the above-mentioned components in the hemolytic agent of the present invention.
  • the buffering agent may, for example, be one selected from the group consisting of phosphoric acid and salts thereof, citric acid and salts thereof, TRIS, and the like, and is usually a buffer system composed of two or more kinds.
  • a metal chelating agent used as an anticoagulant such as sodium EDTA.
  • Osmotic pressure adjusting agents usually inorganic salts such as sodium chloride, sodium sulfate, potassium sulfate, sodium borate, and the like. Preservatives such as isothiazolinone, sodium azide, imidazolidinyl urea.
  • the mixing ratio of the first reagent to the blood sample according to the first embodiment is not particularly limited.
  • the volume mixing ratio of the blood sample to the first reagent may be 1:40 to 1:60.
  • the hemolytic reaction is carried out at a temperature such as 40 to 60 ° C for 15 to 100 seconds, preferably for 40 to 80 seconds. The reaction temperature and time can be adjusted according to specific conditions.
  • deep lysis of red blood cells herein means that the selection of reaction conditions causes the red blood cells to be further lysed as compared with conventional ones, but the platelets can substantially maintain their cell morphology, preferably also White blood cells can maintain their cell morphology.
  • the conventional lysis of red blood cells herein refers to the case where a conventional hemolytic agent is used, and after the reaction, the lysed red blood cells are mixed in the platelet particle group.
  • the method of the first embodiment is capable of causing a deeper degree of lysis of red blood cells, breaking the cell membrane of red blood cells into smaller fragments, and thus obtaining forward and side scatter light by optical detection.
  • the red blood cell debris area and the platelet area can be clearly distinguished, and accurate and accurate detection and counting of platelets can be realized.
  • leukocytes comprising at least mononuclear cells, lymphocytes and neutrophils can be obtained.
  • the blood sample treated with the first reagent is also treated with a second reagent.
  • the second reagent comprises a dye selected from the group consisting of a membrane-specific dye and a mitochondria-specific dye, and/or a nucleic acid-specific dye.
  • the film-specific dye may be selected from one or more of DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, Super Fluor 488, and a deformed structure in which they are parent.
  • the membrane specific dye is Alexa Fluor 488.
  • the mitochondria-specific dye may be selected from the group consisting of Janus Green B, MitoLite Red, Rhodamine 123, and Mitotracker, and one or more of their parent.
  • the mitochondria-specific dye is Mitotracker Deep Red or Mitotracker Red.
  • the deformed structure of the dye includes a commercialized deformed structure or a non-commercially deformed structure.
  • a commercialized deformed structure or a non-commercially deformed structure According to the name, structure, and the like of the dye, those skilled in the art can confirm the deformation of the known dye as the parent from the prior art. Structures (such as commercial deformation structures); at the same time, non-commercial deformation structures can be obtained according to the parent structure and/or existing deformation structures, and it is reasonably expected that these deformation structures can achieve similar dyeing effects to their mother bodies. These deformed structures are all within the scope of the present invention.
  • the "membrane-specific dye” means a fluorescent dye capable of specifically staining a platelet membrane; similarly, “mitochondria-specific dye” means a fluorescent dye capable of specifically staining platelet mitochondria.
  • the platelets and the lysed red blood cells produce a more pronounced fluorescent characteristic, thereby detecting the fluorescence intensity and selecting At least one of the forward scattered light intensity and the side scattered light intensity, particularly by detecting the fluorescence intensity and the forward scattered light intensity, can further distinguish the platelets from the lysed red blood cell fragments.
  • the first reagent in the first embodiment of the present invention contains a surfactant having a high hemolysis ability, so that the fragmentation volume of the red blood cells is smaller.
  • the number of organelle particles scattered from reticulocytes (RET) after hemolysis has a certain correlation with the value of RET. Since reticulocytes are nucleated red blood cells, specific energy can be stained for these particles after the addition of nucleic acid-specific fluorescent dyes.
  • RET reticulocytes
  • the particles may be counted in a preset area of the two-dimensional scattergram of the forward scattered light and the fluorescent signal, and when the count value exceeds a predetermined value (for example, the number of platelets exceeds a certain level), a reticulocyte alarm may be performed to further Subjects were examined.
  • a leukocyte subset of the above three or even four classifications can be obtained and immature granulocytes can be identified.
  • the second reagent may comprise a nucleic acid-specific dye, in particular a nucleic acid-specific dye for reticulocytes.
  • the preferred embodiment can stain the blood sample with a nucleic acid dye, so that not only the information of the reticulocytes can be obtained, but also the platelets can be further differentiated from the organelle particles scattered after the reticulocytes are dissolved.
  • this preferred embodiment of the present invention includes the ability to further achieve an effective measurement of reticulocytes while detecting platelets.
  • these nucleic acid fluorescent dyes can also effectively stain the white blood cell nucleus, and the fluorescent signal can also be used to classify and detect white blood cells.
  • the nucleic acid-specific dye used in the present invention is not particularly limited.
  • Commercially available nucleic acid fluorescent dyes and nucleic acid-specific fluorescent dyes already disclosed in some patent applications can be used in the present invention.
  • the commercially available nucleic acid fluorescent dyes there are SYTO series nucleic acid dyes of Thermofisher Co., Ltd.
  • the fluorescent dye disclosed in Chinese Patent Application No. CN201010022414.6, the anthocyanin dye disclosed in CN200910109215.6, the fluorescent dye disclosed in CN200810216864.1, and the like can be used in the present invention.
  • the entire contents of the above patent documents are hereby incorporated by reference.
  • the concentration range of the nucleic acid dye differs depending on the nature of the dye to be specifically employed, and is not particularly limited, and is usually from 0.002 ppm to 2000 ppm. A preferred concentration range is from 0.03 ppm to 20 ppm.
  • the second reagent preferably further comprises an organic solvent.
  • the organic solvent may be methanol, ethanol, glycerin or the like, but is not limited thereto.
  • the second agent may comprise one of a membrane or a mitochondria-specific dye and one of the nucleic acid-specific dyes to obtain a more accurate and accurate platelet count, and at the same time obtain a differential count of white blood cells and Reticulocyte counts.
  • platelets can be distinguished from other particles in the sample based on information of fluorescence and forward scattered light to obtain a count of platelets (and a mesh obtained in the case of using a nucleic acid dye) The number of erythrocytes is counted), and further information on fluorescence and side scattered light is used to obtain classification information and counts of white blood cells. Further, the intensity of the fluorescence, the forward scattered light, and the side scattered light can be simultaneously utilized to obtain a three-dimensional scattergram of the volume distribution, thereby completing classification and counting of the respective particles.
  • the first embodiment of the second blood detecting method of the present invention detailed above can be all applied to the first blood detecting method of the present invention. Therefore, the first blood detecting method according to the present invention will not be described again.
  • the blood sample is treated with the first reagent and the second reagent to obtain a sample to be tested.
  • the first reagent contains 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 conventional hemolytic agent.
  • An exemplary hemolytic agent such as a quaternary ammonium salt-based cationic surfactant (such as tetradecyltrimethylammonium chloride), but the invention is not limited thereto.
  • the second reagent comprises one selected from the above-described membrane-specific dyes and the above-described mitochondria-specific dyes.
  • the fluorescence characteristics of the platelets and the lysed red blood cells are significantly different, and thus can be detected. Fluorescence intensity and a scattered light intensity, especially by detecting fluorescence intensity and forward scattered light intensity, allow platelets to be well differentiated from lysed red blood cell debris. Due to the use of membrane-specific dyes or mitochondria-specific dyes, in this embodiment, red blood cells can be clearly distinguished from platelets by two-dimensional scattergrams only by lytic lysis, without having to perform red blood cells as in the first embodiment. Deep cracking. Moreover, the protocol can also obtain subpopulations and counts of white blood cells by fluorescence intensity and side scattered light intensity information, and alarm when there are reticulocytes.
  • the second reagent may further comprise a nucleic acid-specific dye.
  • optical information of the reticulocytes can be further obtained therefrom, and preferably counted.
  • the blood detecting method of the present invention can further adopt an optical technique that eliminates interference of a laser to a pulse wave of a platelet optical signal after performing blood cell lysis according to the first embodiment or the second embodiment described above. Detection is performed in the detection system to obtain a more accurate platelet count.
  • Lasers in conventional optical detection systems are susceptible to being affected by reflected light in the optical path and are subject to amplitude variations, frequency shifts, or power spikes.
  • the laser is unstable and oscillates to produce a power spike, its performance on the optical forward signal is a small pulse. These small interfering pulses and the small pulses formed by the platelets in the detection are confused with each other, causing interference.
  • optical detecting system of the present invention will be described in detail with reference to Figs.
  • a magneto-optical device based on the Faraday effect can be used as an optical isolator placed in the optical path.
  • the isolator is mechanically large in size and generally can only be placed outside the light source assembly, and the light beam outside the light source assembly is non-parallel light.
  • the optical isolation effect is severely impaired. Therefore, this solution has the disadvantages of large size, high cost, and poor isolation.
  • an optical detection system includes: an optical subsystem, a flow chamber, and a first detector; the optical subsystem includes: a laser, a front light assembly including an optical isolator, and a backlight including a direct aperture stop Component; among them,
  • the laser configured to emit a laser beam
  • the front light assembly is configured to perform front light processing on the laser beam, and the laser beam processed by the front light is concentrated in the second direction in the direct beam stop, and is concentrated in the first direction
  • the blood cells of the flow cell are measured at the sample and generate scattered light
  • the rear light assembly is disposed after the flow direction of the laser beam, and is configured to perform post-light processing on the scattered light and the laser beam concentrated at the straight stop, so that the light is processed
  • the scattered light of the post-light treatment enters the first detector for light intensity detection;
  • the optical isolator is configured to isolate reflected light generated by the laser beam through the flow chamber and the rear light assembly.
  • FIG. 1 is a schematic structural diagram 1 of an optical detecting system according to an embodiment of the present invention.
  • FIG. 2 is a schematic structural diagram 2 of an optical detecting system 200 according to an embodiment of the present invention. Referring to FIG. 1 and FIG. 2, the embodiment of the present invention is shown in FIG.
  • the optical detection system 200 is provided to include: an optical subsystem 1, a flow chamber 2, a first detector 3;
  • the optical subsystem 1 includes a laser 11, a front light assembly 12 including an optical isolator 121, and a rear light assembly 13 including a direct aperture 131;
  • the laser 11 is configured to emit a laser beam
  • the front light assembly 12 is configured to perform a front light treatment on the laser beam, and the laser beam processed by the front light is concentrated in the second direction at the direct beam stop 131, and is concentrated in the first direction.
  • the blood cells of the flow chamber 2 are tested at the sample and generate scattered light;
  • the rear light assembly 13 is disposed behind the flow chamber 2 along the propagation direction of the laser beam, and is configured to perform post-light processing on the scattered light and the laser beam concentrated at the straight stop 131. And causing the scattered light processed by the post-light to enter the first detector 3 to perform light intensity detection;
  • the optical isolator 121 is configured to isolate reflected light generated by the laser beam through the flow chamber and the rear light assembly.
  • the laser 11 in the optical subsystem is described.
  • the laser 11 is a semiconductor laser, and in actual implementation, it may be a P-line polarized laser; in practical applications, the wavelength of the laser beam emitted by the laser is determined.
  • the design of the main parameters of the optical path, such as the selection of the lens, the selection of the signal collection angle, etc., and the wavelength of the laser beam is also related to the reagents used in the detection, such as fluorescent dyes.
  • the laser emitted by the laser 11 The wavelength of the light beam is 630 nm to 640 nm.
  • the optical isolator 121 in the front light assembly 12 is composed of a beam splitting prism and a polarization state conversion component which are connected to each other by adhesion;
  • the beam splitting prism is configured to reflect an S-polarized component of the incident laser beam and transmit a P-polarized component of the incident laser beam;
  • the polarization state conversion component is configured to change a polarization state of the P polarization component transmitted through the beam splitting prism, change the P polarization component from linearly polarized light to circularly polarized light, and change the circularly polarized light reflection
  • the latter polarization state is S-polarized light and is reflected by the beam splitting prism.
  • FIG. 3 is a schematic diagram of the principle of the optical isolator provided by the embodiment of the present invention.
  • the laser beam L TM mode
  • the laser The P-polarized light 31 in the light beam L i.e., the polarization component of the parallel paper surface
  • the S-polarized light 32 i.e., the polarization component of the vertical paper surface
  • the P-polarized light 31 passes through the 1/4 wave plate 36, and the polarization state of the P-polarized light 31 after passing through the 1/4 wave plate 36 changes, from linearly polarized light to circularly polarized light 33; circular polarization
  • the reflected light of the light 33 passes through the 1/4 wavelength plate 36 again, the polarization state changes, and the circularly polarized light is converted into the S-polarized light 34, and the S-polarized light 34 is The 45° slope of the dichroic prism 35 is reflected out and is not fed back to the laser, thereby achieving isolation of the reflected light from the optical path.
  • FIG. 4 is a schematic diagram of a first incident surface of the optical isolator according to an embodiment of the present invention, wherein the first incident surface is indicated by reference numeral 41, and in actual implementation, the first The light reflectivity of the incident surface can be achieved by the coating design and process of the first incident surface.
  • the optical isolator 121 can also be composed of an analyzer and a polarization conversion component that are connected to each other by bonding;
  • the analyzer is configured to allow only a P-polarized component of the laser beam to pass;
  • the polarization state conversion component is configured to change a polarization state of the P-polarized component of the analyzer, to change the P-polarization component from linearly polarized light to circularly polarized light, and to change the circularly polarized light reflection
  • the polarization state of the reflected light is S-polarized light and is isolated by the analyzer.
  • the polarization conversion component as a magneto-optical crystal as an example
  • the laser beam is incident on the analyzer
  • only the P-polarized light in the laser beam can pass through the analyzer, and the P-polarized light passing through the analyzer enters the magneto-optical crystal.
  • the polarization state of the P-polarized light passing through the magneto-optical crystal changes, and its polarization direction is rotated by 45°.
  • the polarization direction is rotated by 45° again to form S-polarized light perpendicular to the polarization state of the original P-polarized light, which is isolated by the analyzer and does not feed back.
  • the polarization state conversion component may also be an optically active crystal.
  • the beam splitting prism and the analyzer may be combined with any one of a quarter wave plate, a magneto-optical crystal, and an optical crystal to realize an optical path. The isolation of reflected light.
  • the optical isolator 121 is composed of a band pass filter and a frequency doubling crystal which are connected to each other by bonding;
  • the band pass filter is configured to pass the laser beam having a wavelength of ⁇ ;
  • the frequency doubling crystal is configured to multiply a laser beam passing through the band pass filter, and multiply the reflected light of the multiplied laser beam by the band pass filter The pieces are filtered out.
  • the optical isolator has an optical isolation of not less than 30 db.
  • the front light assembly 12 further includes a collimating lens 122;
  • the collimating lens 122 is disposed between the laser 11 and the optical isolator 121 along a propagation direction (optical axis direction) of the laser beam, and is configured to perform collimation processing on the laser beam.
  • the laser beam becomes a parallel beam.
  • the front light assembly 12 further includes a first light concentrating component 123 and a second light concentrating component 124;
  • the first light concentrating component 123 is configured to perform a first focusing on the laser beam, so that the laser beam converges in a first direction at a blood cell sample to be measured in the flow chamber and generates scattered light;
  • the second light concentrating component 124 is configured to perform a second focusing on the laser beam such that the laser beam converges in the second direction at the straight beam 131.
  • the second direction is a lateral direction, that is, a direction perpendicular to a flow direction of the blood cell sample to be tested;
  • the first direction is a longitudinal direction, that is, the flow direction of the blood cell sample to be tested is the same The direction.
  • the first light concentrating component 123 and the second light concentrating component 124 may be implemented by different cylindrical mirrors of optical parameters (such as focal length, etc.), for example, the first light concentrating component 123 is implemented by a first cylindrical mirror, The two-light converging assembly 124 is implemented by a second cylindrical mirror.
  • the second light concentrating component 124 is taken as an example for the second cylindrical mirror.
  • FIG. 5 is a schematic diagram of the light concentrating principle of the second cylindrical mirror according to the embodiment of the present invention.
  • the laser beam passes through the second cylindrical mirror.
  • the second cylindrical mirror does not process the laser beam in the longitudinal direction, and the laser beam passing through the second cylindrical mirror is compressed in the lateral direction, and the laser beam is focused in the lateral direction (perpendicular to the flow direction of the blood cell sample to be tested).
  • the focus is on the straight pupil.
  • FIG. 6 is a front view of the straight-line diaphragm provided by the embodiment of the present invention, and the laser beam irradiated to the blood cell sample in the flow chamber is scattered.
  • the scattered light is collected by the straight-line diaphragm.
  • the straight-line diaphragm constrains the collection angle of the low-angle scattering signal; at the same time, the straight-line diaphragm converges in the second direction at the laser beam blocking the direct aperture. The beam is blocked.
  • the light collecting angle of the straight stop may be 1 to 10 degrees.
  • the rear light assembly 13 further includes a third convergence assembly 132 and a small aperture stop 133;
  • the third converging component 132 is configured to perform third focusing on the scattered light, converge the scattered light at the aperture stop, and enter the first hole through the aperture of the aperture aperture A detector for light intensity detection.
  • the third convergence component can be one of the following:
  • a lens group comprising at least one plano-convex lens and at least one lenticular lens
  • a lens group composed of at least two plano-convex lenses
  • a lens group composed of at least two lenticular lenses
  • a lens group composed of at least two spherical lenses
  • the optical detection system further includes a second detector 4 and a fluorescence detector 5;
  • the second detector is disposed in a direction that is within a set angle range from an angle of propagation of the laser beam, and is configured to be at a set angle range with respect to a direction of propagation of the laser beam.
  • the scattered light inside is detected by light intensity
  • the fluorescence detector is disposed in a direction within a set angle range from an angle of propagation of the laser beam, and is configured to perform fluorescence detection on fluorescence generated by the blood cell sample to be tested.
  • FIG. 7 is a schematic structural diagram of a blood analysis system according to an embodiment of the present invention.
  • the blood analysis system according to the present invention generally includes a sampling portion, a reagent supply portion, a reaction portion, an optical detection system, and a data processing module.
  • FIG. 7 shows a specific blood analysis system in accordance with the present invention.
  • the blood analysis system specifically includes a first cabinet 100, a second cabinet 200, a sampling unit 10, a reagent supply unit (not shown), a reaction unit 30, an optical detection system 50, a data processing module 70, and an output unit 90.
  • the output portion 90 can be a user interface.
  • the optical detection system 50 and the data processing module 70 are disposed inside the second casing 200 and are respectively disposed on two sides of the second casing 200.
  • the reaction portion 30 is disposed inside the first casing 100, and the output portion 90 and the sampling portion 10 are on the outer surface of the first casing 100.
  • the sampling unit 10 has a sampling needle, collects a blood sample, and delivers the collected blood sample to the reaction unit 30.
  • the reagent supply portion stores a reagent for reacting with the blood sample (for example, at least the aforementioned first reagent is stored) and supplies the corresponding reagent to the reaction portion as needed.
  • the reaction portion 30 is configured to react the blood sample from the sampling portion and the reagent from the reagent supply portion to obtain a liquid to be tested containing a plurality of the platelet particles, so that the platelet particles are optically detected first-by-one The flow chamber of the system.
  • the optical detection system 50 includes an optical subsystem, a flow chamber, and a first detector.
  • the optical subsystem includes: a laser, a front light assembly, and a rear light assembly, the front light assembly including an optical isolator; wherein the laser emits a laser beam; the front light assembly is configured to face the laser beam Performing a front light treatment, the laser beam irradiated by the front light is concentrated in a first direction at a blood particle (such as a platelet particle) of the flow chamber and generates scattered light; the backlight assembly is along the laser beam a direction of propagation disposed after the flow chamber, configured to post-process the scattered light such that the scattered light processed by the post-light enters the first detector for light intensity detection; An isolator configured to isolate reflected light from the laser; the reflected light being generated by the laser beam through the flow chamber and the rear light assembly.
  • the flow chamber is for queuing blood particles such as platelet particles.
  • the first detector is used to detect optical information, in particular light intensity information,
  • the data processing module 70 is configured to detect blood (eg, platelet) particles flowing through the flow chamber according to the light intensity signal of the scattered light detected by the first detector to obtain corresponding blood particles. Test results.
  • blood eg, platelet
  • the output unit 90 is configured to output a detection result corresponding to the blood (eg, platelet) particles.
  • FIG. 8 is a schematic structural diagram of an optical detection system according to an embodiment of the present invention.
  • an optical detection system according to an embodiment of the present invention includes a laser 71, a front light assembly 72, a flow chamber 73, and a rear light assembly 74.
  • blood particles especially in the form of platelet particles
  • the flow direction of the platelet particles is perpendicular to the paper surface direction.
  • the laser beam emitted by the laser 71 travels in a direction parallel to the plane of the paper.
  • the laser 71 is a P-line polarized laser, and the laser beam 71 emits a laser beam having a wavelength of 630 nm to 640 nm.
  • the front light assembly 72 includes a collimating lens 721, an optical isolator 722, a first cylindrical mirror 723, and a second cylindrical mirror 724 which are sequentially disposed along the propagation direction of the laser beam;
  • the rear light assembly 74 includes along The straight beam stop 741, the plano-convex lens 742, the lenticular lens 743, and the aperture stop 744 are sequentially disposed in the propagation direction of the laser beam.
  • the laser beam emitted by the laser 71 is collimated by the collimating lens 721 to form a parallel laser beam, which is then transmitted through the optical isolator 722 and focused on the flow chamber in the longitudinal direction (perpendicular to the plane of the paper) via the first cylindrical mirror 723.
  • the center of 73 is used as a detecting spot, and the angle between the direction of propagation of the laser beam and the light incident surface of the flow chamber is at right angle, and the laser beam focused on the flow chamber is irradiated to the platelet particles in the flow chamber to generate light scattering.
  • the size of the laser beam in the longitudinal direction matches the size of the platelet particles, and the laser beam passing through the first cylindrical mirror 723 enters the second cylindrical mirror 724 in the lateral direction ( It is compressed parallel to the paper surface and converges in the lateral direction at the straight stop 741.
  • the scattered light generated by the platelet particles irradiated into the flow chamber is collected by the straight-line diaphragm, and then focused by the lens group composed of the plano-convex lens 742 and the lenticular lens 743, and concentrated at the small aperture stop 744, and the small aperture light is received.
  • the aperture of the ⁇ 744 enters the forward detector 75, and the forward detector 75 performs forward optical signal strength detection.
  • the lateral detector 76 and the fluorescence detector 77 are disposed in a direction perpendicular to the propagation direction of the laser beam; wherein the lateral detector 76 is configured to oppose the laser beam The light is detected by the vertical scattered light in the propagation direction; the fluorescence detector 77 is disposed in a direction perpendicular to the propagation direction of the laser beam, and is configured to perform fluorescence detection on the scattered light.
  • the laser beam emitted by the laser 71 propagates in the optical path shown in FIG. 8 to form reflected light
  • the optical isolator 722 is configured to isolate the reflected light generated by the laser beam emitted from the laser 71 in the optical path.
  • the optical isolator 722 is composed of a beam splitting prism and a quarter wave plate which are connected to each other by adhesion;
  • the beam splitting prism is configured to reflect an S-polarized component of the incident laser beam and transmit a P-polarized component of the incident laser beam;
  • the quarter-wave plate is configured to change a polarization state of the P-polarized component transmitted through the spectroscopic prism, to change the P-polarized component from linearly polarized light to circularly polarized light, and to change the circularly polarized light
  • the reflected polarization state is S-polarized light and is reflected by the beam splitting prism.
  • the P-polarized light in the laser beam ie, the polarization component of the parallel paper surface
  • the S-polarized light ie, the polarization component of the vertical paper surface
  • the 45° slope is reflected out; then, the P-polarized light passes through the 1/4 wave plate, and the polarization state of the P-polarized light after the 1/4 wave plate changes, from linearly polarized light to circularly polarized light; circularly polarized light After the reflected light (reflected light reflected by the subsequent optical path) passes through the 1/4 wave plate again, the polarization state changes, and the circularly polarized light is converted into S-polarized light, and the S-polarized light is split by the 45° bevel of the dichroic prism. Reflected out, it will not be fed back to the laser, thus achieving the isolation of the reflected light from the optical path.
  • FIG. 4 is a schematic diagram of a first incident surface of the optical isolator according to an embodiment of the present invention, wherein the surface indicated by reference numeral 41 is the first incident surface, and in actual implementation, The light reflectivity of an incident surface can be achieved by the coating design and process of the first incident surface.
  • the optical isolator 121 may also be composed of an analyzer and a polarization conversion component that are connected to each other by bonding;
  • the analyzer is configured to allow only a P-polarized component of the laser beam to pass;
  • the magneto-optical crystal is configured to change a polarization state of the P-polarized component passing through the analyzer to rotate its polarization direction by 45°. After the reflected light, which is reflected by the subsequent optical path, passes through the magneto-optical crystal again, the polarization direction is again rotated by 45° to form S-polarized light perpendicular to the polarization state of the original P-polarized light, thereby being isolated by the analyzer.
  • the analyzer When the laser beam is incident on the analyzer, only the P-polarized light in the laser beam can pass through the analyzer, and the P-polarized light passing through the analyzer enters the magneto-optical crystal, and the polarization state of the P-polarized light passing through the magneto-optical crystal changes. Its polarization direction is rotated by 45°. After the reflected light, which is reflected by the subsequent optical path, passes through the magneto-optical crystal again, the polarization direction is rotated by 45° again to form S-polarized light perpendicular to the polarization state of the original P-polarized light, thereby being isolated by the analyzer. Will not give back to the laser.
  • the optical isolator may also be composed of a band pass filter and a frequency doubling crystal which are connected to each other by bonding;
  • the band pass filter is configured to pass the laser beam having a wavelength of ⁇ ;
  • the frequency doubling crystal is configured to multiply a laser beam passing through the band pass filter, and multiply the reflected light of the multiplied laser beam by the band pass filter The pieces are filtered out.
  • the optical isolator has an optical isolation of not less than 30 db.
  • the angle between the propagation direction of the laser beam and the light incident surface of the flow chamber may be a non-right angle, that is, the position of the flow chamber in the above embodiment is performed on the xy plane or the paper surface. The deflection of the angle is such that the flow chamber is placed obliquely.
  • FIG. 9 is a schematic structural diagram of the optical detection system according to the embodiment of the present invention. As shown in FIG. 9, the propagation direction of the laser beam and the light incident surface of the flow chamber are shown. The angle ⁇ between the two is an acute angle. In one embodiment, the angle ⁇ ranges from 80° to 90°.
  • the light beam is reflected after the surface of the flow chamber is reflected. Deviation from the optical axis (laser beam propagation direction), thereby reducing the luminous flux of the reflected light entering the laser.
  • the present application can be applied as long as the reflected light from the light incident surface of the flow cell does not enter the angle ⁇ of the front light assembly.
  • the platelet particles are small and flow through the flow chamber to form small pulses.
  • the optical isolator in the optical detection system of the above blood cell analyzer can well isolate the reflected light generated when the laser beam propagates in the optical path.
  • the laser can stably output the laser beam, avoiding the small pulse of the power spike generated by the reflected light entering the laser, that is, avoiding the interference of the small pulse and the small pulse formed by the platelet particle, thereby greatly improving the detection precision.
  • An embodiment of the present invention further provides a method for detecting a platelet, which is applied to the above blood cell analyzer including an optical detection system, the optical detection system comprising: a laser, a front light component including an optical isolator, a backlight assembly, a flow chamber, and a first detector; the method comprising:
  • the incoming light is detected by the first detector using the first detector to obtain a first detection result to identify the platelet particles based on the first detection result.
  • the method further includes:
  • the collected blood sample is subjected to a hemolysis treatment such that red blood cells in the blood sample are cleaved to obtain a liquid to be tested containing a plurality of the platelet particles.
  • the method further includes:
  • the laser beam is pre-processed using a front light assembly such that the laser beam processed by the front light converges in a second direction at a straight stop included in the rear light assembly.
  • the blood cell analyzer further includes a second detector; and correspondingly, the method further includes:
  • the second detector uses the second detector to perform light intensity detection on the scattered light that is within a set angular range with respect to the propagation direction of the laser beam, to obtain a second detection result, in combination with the first detection result and the The second detection result identifies the platelet particles.
  • the blood cell analyzer further includes a fluorescence detector; and correspondingly, the method further includes:
  • the platelet particles in the liquid to be tested are dyed by a specific fluorescent dye;
  • the specific fluorescent dye may be at least one of a film dye and a mitochondrial dye;
  • the platelet particles in the sample to be tested also generate fluorescence when passing through the detection zone, and the fluorescence detector is configured to detect fluorescence generated by the platelet particles to obtain a third detection result to be combined with the The first detection result and the third detection result identify the platelet particles.
  • the blood cell analyzer further includes a second detector and a fluorescence detector; and correspondingly, the method further includes:
  • the platelet particles in the liquid to be tested are subjected to fluorescent staining treatment
  • the second detector is configured to perform light intensity detection on the scattered light within an angle range set by the angle of propagation of the laser beam to obtain a second detection result; wherein the set angle is The range can be 60°120°.
  • the fluorescence detector is configured to detect fluorescence generated by the platelet particles to obtain a third detection result
  • the platelet particles and white blood cell count and classification are identified in conjunction with the first detection result, the second detection result, and the third detection result.
  • the reflected light generated by the laser beam propagating in the optical path can be well isolated, so that the laser can stably output the laser beam, thereby avoiding the small pulse of the power spike generated by the reflected light entering the laser, and avoiding the
  • the small pulse that interferes with the formation of small pulses and platelet particles is confused with each other, which greatly improves the detection accuracy of the blood cell analyzer.
  • the hemolytic agent of the present invention is formulated in the following formulation.
  • the white blood cell and PLT concentrations of the sample can be calculated to be 9.8 ⁇ 10 9 /L and 166 ⁇ 10 9 /L, respectively.
  • the concentration of white blood cells was measured by Beckman particle counter Z2 to be 9.6 ⁇ 10 9 /L; the concentration of PLT was 171 ⁇ 10 9 /L by manual microscopic examination.
  • the platelet and white blood cell counts obtained by the method of the present invention are in good agreement with the results of the conventional methods.
  • the detection reagent of the present invention was formulated in the following formulation.
  • Example 3 Deep hemolysis and addition of nucleic acid dyes for platelet and white blood cell counting by lateral light scattering and fluorescent signals
  • the detection reagent of the present invention was formulated in the following formulation.
  • the addition of the nucleic acid dye can effectively distinguish the platelets and RET in the fluorescence direction.
  • the PLT concentration of the sample can be calculated to be 198 ⁇ 10 9 /L.
  • the PLT concentration calculated by the manual microscopic examination method was 202 ⁇ 10 9 /L.
  • the nucleic acid staining method of the present embodiment can effectively distinguish platelets from red blood cell fragments.
  • the method of the present example has no effect on the sorting count of white blood cells.
  • Another 20 ⁇ l of the same blood sample was subjected to the same treatment as above, and measured in the same flow cytometer, the excitation wavelength was set to 488 nm, the gain was 100,000, and the 90-degree lateral fluorescence intensity information and the 90-degree side scattered light intensity were collected. Information, a cell scatter plot is obtained, as shown in Figure 12B.
  • the dye is also effective for staining white blood cells while staining PLT, and can be clearly grouped.
  • the WBC concentration of the sample can be calculated to be 7.86 ⁇ 10 9 /L.
  • the WBC concentration measured on the same sample by the conventional reference method in combination with the shellman particle counter depth Z2 was 8.01 x 10 9 /L. There is good agreement between the two methods.
  • the ratios were 21.2%, 3.5%, 74.8%, and 0.5%, respectively.
  • the ratios of lymphocytes, monocytes, neutrophils, basophils, and eosinophils were 20.8%, 3.2%, and 75.1%, respectively. And 0.9%.
  • the method of the present invention has a good correlation between the division of white blood cells and the ratio obtained by a commercially available blood cell analyzer.
  • the detection reagent of the present invention was formulated in the following formulation.
  • Fluorescent dye structure
  • RET and PLT can be clearly distinguished by fluorescence and forward scattered light signals, respectively.
  • the PLT concentration of the sample was calculated to be 165 ⁇ 10 9 /L, and the concentration of RET was 18.8 ⁇ 10 9 /L.
  • the value of PLT was 175 ⁇ 10 9 /L and the concentration of RET was 20.1 ⁇ 10 9 /L by a conventional artificial mirror. It is indicated that the method of the present invention can effectively distinguish and count PLT and RET in blood samples.
  • white blood cells can be clearly classified into lymphocytes, monocytes, neutrophils and basophils, and eosinophils.
  • the WBC concentration of the sample can be calculated to be 8.32 ⁇ 10 9 /L.
  • the WBC concentration measured for the same sample by the conventional reference method in combination with the shellman particle counter Z2 was 8.45 x 10 9 /L. It can be seen that there is good consistency between the two methods.
  • the ratios were 15.1%, 6.1%, 75.5%, 2.3%, and 1.0%, respectively.
  • the proportions of lymphocytes, monocytes, neutrophils and basophils, eosinophils and immature granulocytes were 14.8% and 5.7%, respectively. 76.2%, 1.9% and 1.4%. From the above results, it can be seen that the method of the present invention has a good correlation between the division of leukocytes and the ratio obtained by a commercially available blood cell analyzer.
  • Example 5 Deep hemolysis and addition of nucleic acid dyes to detect platelets and white blood cells by light scattering and fluorescent signals
  • the detection reagent of the present invention was formulated in the following formulation.
  • Fluorescent dye structure
  • RET and PLT can be clearly distinguished by the fluorescence signal and the forward scattered light signal, respectively.
  • the PLT concentration of the sample was calculated to be 189 ⁇ 10 9 /L, and the concentration of RET was 101 ⁇ 10 9 /L.
  • the value of PLT detected by a conventional artificial mirror was 201 ⁇ 10 9 /L, and the concentration of RET was 112 ⁇ 10 9 /L. It is indicated that the method of the present invention can effectively distinguish and count PLT and RET in blood samples.
  • White blood cells can be clearly divided into lymphocytes, monocytes, neutrophils and basophils, and eosinophils.
  • the WBC concentration of the sample can be calculated to be 6.54 ⁇ 10 9 /L.
  • the WBC concentration measured for the same sample by the conventional reference method in combination with the shellman particle counter Z2 was 6.32 x 10 9 /L. It can be seen that there is good consistency between the two methods.
  • the ratios of eosinophils were 19.8%, 3.2%, 75.8%, and 1.2%, respectively.
  • the proportions of lymphocytes, monocytes, neutrophils, basophils, and eosinophils were 20.3%, 2.9%, and 75.1%, respectively. 1.7%. From the above results, it can be seen that the method of the present invention has a good correlation between the division of white blood cells and the ratio obtained by a commercially available blood cell analyzer.
  • Example 6 Deep hemolysis and addition of membrane dyes to detect platelets and white blood cells by light scattering and fluorescent signals
  • the detection reagent of the present invention was formulated in the following formulation.
  • Fig. 15A both dark clusters (bottom and right) are lysed red blood cell fragments, and the middle cluster is platelets, indicating that the method can also clearly distinguish platelets from red blood cell fragments.
  • the samples were tested on the Mind 6800 hematology analyzer by dividing the ratio of lymphocytes, monocytes, neutrophils and eosinophils to 16.4%, 6.1%, and 77.5%, respectively. After that, the ratios of lymphocytes, monocytes, neutrophils, and eosinophils were 16%, 5.8%, 78.2%, and 1.9%, respectively.
  • the method of the present invention has a good correlation between the division of white blood cells and the ratio obtained by a commercially available blood cell analyzer.
  • Example 7 Deep hemolysis and addition of mitochondrial dyes to detect platelets and white blood cells by light scattering and fluorescent signals
  • the detection reagent of the present invention was formulated in the following formulation.
  • Example 8 Deep hemolysis and addition of membrane dyes and nucleic acid dyes to detect platelets and reticulocytes by light scattering and fluorescent signals
  • the detection reagent of the present invention was formulated in the following formulation.
  • both dark clusters are lysed red blood cell fragments, and the middle cluster is platelets, indicating that the method can more clearly distinguish platelets from red blood cell fragments; the rightmost cluster identification In order to reticulate red blood cells, the method can further obtain information on reticulocytes.
  • the samples were tested on the Mind 6800 hematology analyzer by dividing the ratio of lymphocytes, monocytes, neutrophils and eosinophils to 16.8%, 6.4%, and 76.8%, respectively. After that, the ratios of lymphocytes, monocytes, neutrophils, and eosinophils were 16%, 5.8%, 78.2%, and 1.9%, respectively.
  • the method of the present invention has a good correlation between the division of white blood cells and the ratio obtained by a commercially available blood cell analyzer.
  • the detection reagent of the present invention was formulated in the following formulation.
  • Fluorescent dye structure
  • RET and PLT can be distinguished by a fluorescent signal and a forward scattered light signal, respectively.
  • FIG. 18A and B shows:
  • the nucleic acid dye can distinguish and stain PLT and RET in deep hemolysis state, and can count related particles.
  • the test results are well correlated.
  • Fig. 19A when the membrane dye Alexa Fluor 488 is used, not only the platelets (in the circle) and the blood shadow can be effectively distinguished, but also the dyeing effect on the white blood cells is good, and the white blood cells can be realized.
  • the platelet value of the sample was calculated to be 216 ⁇ 10 9 /L by the volume of the injection volume and the number of test particles of the platelet, and the measurement value by the classical artificial microscopic examination method was also 216 ⁇ 10 9 /L.
  • the white blood cells were counted using the same method, and the result was 8.86 ⁇ 10 9 /L, and the value was 8.81 ⁇ 10 9 /L by the Beckman particle counter Z2, which had good consistency.
  • the proportion of lymphocytes, monocytes, neutrophils, and eosinophils was 17.8%, 4.8%, 75.8, and 1.6%, respectively, and the samples were tested on a Mindray 6800 hematology analyzer.
  • the ratios of monocytes, neutrophils, and basophils and eosinophils were 18.1%, 5.1%, 74.8%, and 2.0%, respectively. From the above results, it can be seen that the above methods divide white blood cells.
  • the ratio in the blood cell analyzer has a good correlation.
  • Fig. 20A It can be seen from Fig. 20A that after the dye is dyed, the platelets and the blood shadow can be effectively distinguished, and the scatter number is divided, and the platelet concentration is calculated to be 158 ⁇ 10 9 /L, and the classic artificial mirror is passed.
  • the measurement method of the detection method is 160 ⁇ 10 9 /L, and therefore, the dye can effectively distinguish and count the platelet particles in the hemolysis state.
  • the white blood cells were counted using the same method, and the result was 4.35 ⁇ 10 9 /L, and the measured value of the Beckman particle counter Z2 by the reference method was 4.28 ⁇ 10 9 /L, both of which had better consistency.
  • the proportion of lymphocytes, monocytes, neutrophils, and eosinophils was 14.8%, 6.8%, 74.5%, and 3.9%, respectively, and the sample was tested on a Mindray 6800 hematology analyzer.
  • the proportions of cells, monocytes, neutrophils, and eosinophils were 15.9%, 7.2%, 72.2%, and 4.7%, respectively. From the above results, it can be seen that the above methods divide white blood cells and blood cell analyzers. The ratio has a good correlation.
  • Fig. 21A It can be seen from Fig. 21A that after dyeing Rhodamine 123, platelets and blood shadows can be effectively distinguished, and the number of scatter points is divided, and the platelet concentration is calculated to be 208 ⁇ 10 9 /L.
  • the measurement method of the classical artificial microscopic examination method is 201 ⁇ 10 9 /L. Therefore, the dye rhodamine 123 can effectively distinguish and count the platelet particles in the hemolysis state.
  • the white blood cells were counted using the same method, and the result was 4.02 ⁇ 10 9 /L, and the measured value of the Beckman particle counter Z2 by the reference method was 3.98 ⁇ 10 9 /L, which was better. consistency.
  • the proportion of lymphocytes, monocytes, neutrophils, and eosinophils was 15.9%, 7.1%, 71.5%, and 5.5%, respectively, and the sample was tested on a Mindray 6800 blood cell analyzer.
  • the proportions of cells, monocytes, neutrophils, and eosinophils were 15.9%, 6.8%, 72.2%, and 5.1%, respectively. From the above results, it can be seen that the above methods divide white blood cells and blood cell analyzers. The ratio has a good correlation.
  • Fig. 22A when Mitotracker Deep Red is used, platelets (the range of the dotted line in the figure) can be distinguished from the blood shadow, wherein the light dispersion point is expressed as platelets, and the number of scatter points is divided to calculate
  • the concentration of PLT was 165 ⁇ 10 9 /L, and it was counted by classical artificial microscopy. The concentration was 201 ⁇ 10 9 /L. It can be seen that the dye Mitotracker Deep Red can also distinguish platelets.
  • the white blood cells were counted using the same method, and the result was 5.12 ⁇ 10 9 /L, and the measured value of the Beckman particle counter Z2 by the reference method was 5.08 ⁇ 10 9 /L, which was better. consistency.
  • the proportion of lymphocytes, monocytes, neutrophils, and eosinophils was 14.8%, 7.8%, 74.5%, and 2.9%, respectively, and the sample was tested on a Mindray 6800 blood cell analyzer.
  • the proportions of cells, monocytes, neutrophils, and eosinophils were 15.9%, 7.2%, 73.2%, and 3.7%, respectively. From the above results, it can be seen that the above methods divide white blood cells and blood cell analyzers. The ratio has a good correlation.
  • Fig. 23A when the dye Mitotracker Red is used, not only the platelets (the dotted line in the figure) and the blood shadow can be distinguished more effectively, but also from Fig. 23B, it can be seen that the dyeing performance of the dye on the white blood cells is also It can effectively classify white blood cells, thereby realizing the effective classification and counting of platelets while detecting white blood cells.
  • the platelet value of the sample was calculated to be 176 ⁇ 10 9 /L by the volume of the injection volume and the number of test particles of the platelet, and the measurement value by the classical artificial microscopic examination was 172 ⁇ 10 9 /L, which was better. Consistency.
  • the white blood cells were counted using the same method, and the result was 7.65 ⁇ 10 9 /L, and the measured value of the Beckman particle counter Z2 by the reference method was 7.73 ⁇ 10 9 /L, both of which had better consistency.
  • the proportions of lymphocytes, monocytes, neutrophils, and eosinophils and immature granulocytes were 13.7%, 6.1%, 77.5%, 0.7%, and 2.0%, respectively, on the Mindray 6800 Hematology Analyzer.
  • the proportions of lymphocytes, monocytes, neutrophils, eosinophils and immature granulocytes were 14.1%, 5.8%, 76.2%, 1.9% and 2.0%, respectively.
  • the above method has a good correlation with the division of white blood cells with the ratio in the blood cell analyzer.
  • Example 15 uses Example 10 as an example to verify the correlation of platelet test
  • the test method for each blood sample is as follows: Take 20 ⁇ l of blood sample and add it to 1 mL of the above-mentioned first reagent solution, and add 20 ⁇ l of the second reagent, and incubate at 35 ° C for 60 seconds.
  • the data collected by flow cytometry (Mirray BriCyte E6) (excitation wavelength 488 nm) was set to a gain of 500, and a 90-degree lateral fluorescence signal was collected to collect fluorescence staining information, and forward-scattering light intensity information with an angle of 0 was measured.
  • flow cytometry Mirray BriCyte E6
  • excitation wavelength 488 nm excitation wavelength 488 nm
  • test values are in good agreement with the Mind 6800 instrument test values, ranging from 100 ⁇ 10 9 /L to 250 ⁇ 10 9 /L. Within the range of values, the correlation coefficient is 0.9817.

Abstract

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，该第一试剂包括溶血剂，该溶血剂将该血液样本中的红细胞裂解为其光散射特性显著不同于血小板的碎片；使待测试样中的粒子逐个通过光学检测系统的检测区，获取该待测试样的光学信息；和根据该光学信息中的至少两种获得血小板的光学信息。该方法通过裂解血液样本中的红细胞获取血小板计数，并可同时区分白细胞亚群。

Description

一种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，血小板的常用测定方法之一是电阻抗法。该方法是使含血细胞的样本从具有两个电极的小孔间通过，当有血细胞(例如血小板)通过时，电阻发生变化，从而产生电阻脉冲，然后将检测到的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。
针对这种情况，已提出了用对血小板的表面抗原有特异性的标记抗体对血小板进行标记并计数的方法(美国临床病理学杂志(2001)：115，p460-464)。该方法，由于在测试过程中需要使用抗原抗体反应，因而得到结果需要花很长时间。因此，该方法不适用于诸如判断是否需要输血等需紧急作出判断的测定。而且该方法中的检测试剂也相对昂贵。
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。但是在血液中有时会出现破碎的红细胞和脂质等，其大小与血小板类似，也同样被染色，因此成为影响血小板测定的杂质。特别是在测定需要输血的血小板低的样本时，这些杂质的影响会更大。
针对这种情况，SYSMEX公司的中国专利申请公开CN 101173921中公开了一种特异性染色剂，能够在荧光方向有效地从其他血细胞和脂质颗粒等杂质中区分出血小板。但这种方法需要在单独的测试通道中进行，并且这种染色剂仍不能对红细胞碎片和血小板进行有效的区分。
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，本发明的目的是提供一种新颖的血液检测方法，该方法利用经溶血的试样，并根据光学信息就能够准确检测血液样本中的血小板。
本发明进一步的目的是在上述检测方法中，根据光学信息进一步检测血液中白细胞，以及更进一步的报警网织红细胞或检测网织红细胞。
本发明的另一目的是提供实施上述方法的血液检测系统。
为实现上述目的，本发明首先提供第一种血液检测方法，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其光散射特性显著不同于血小板的碎片；
使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信 息；和
根据所述光学信息中的至少两种获得血小板的光学信息。
根据一种实施方式，所述至少两种光学信息为前向散射光强度和侧向散射光强度，以将血小板与裂解的红细胞碎片区分开。
在该实施方式中，所述方法进一步包括根据前向散射光强度和侧向散射光强度的光学信息获得白细胞的光学信息，优选地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。
具体地，所述溶血剂为选自烷基糖苷、三萜皂苷、甾族皂苷中的至少一种。
根据优选的实施方式，所述烷基糖苷选自具有通式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。
所述单糖可选自五碳糖、甲基五碳糖和六碳糖，其中五碳糖诸如阿拉伯糖、木糖、核糖、来苏糖等；甲基五碳糖诸如夫糖、鼠李糖、鸡那糖等；六碳糖诸如葡萄糖、甘露糖、果糖、半乳糖、山梨糖；所述去氧单糖，诸如去氧核糖、去氧葡萄糖等；所述多糖诸如麦芽糖、蔗糖等。
n优选为6～14的整数，更优选为7～11的整数。
进一步优选，所述第一试剂进一步包括：
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 PCTCN2019084660-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。
所述通式II的非离子型表面活性剂中，优选地，R 1为C8-C18的直链烷基。C8-C18的直链烷基具体可为辛基、葵基、月桂基、十四烷基、十六烷基或硬脂基。更优选R 1为C12-C16的直链烷基，具体可为月桂基、十四烷基或十六烷基。R 2优选为-O-。m优选为15～30。
该实施方式中，所述第一试剂优选含有0.025g/L～10g/L、优选0.1g/L～5.0g/L的具有通式I的糖苷类化合物，和0.03～1.5g/L、优选0.05～1.0g/L的具有通式II的非离子型表面活性剂。
所述第一试剂还可包括选自缓冲剂、金属螯合剂、渗透压调节剂和防腐剂中的一种或多种。
在一种具体的实施方式中，所述血液样本与所述第一试剂的体积混合比可为1:40～1:60。用所述第一试剂在诸如40～60℃的温度下，反应15～100秒，优选反应40～80秒来处理所述血液样本。
根据该实施方式，所述试样中的红细胞经第一试剂处理后被深度裂解，而血小板仍保持其细胞形态，这样所得红细胞碎片的散射光性质与血小板的散射光性质出现了显著差异。由前向散射光和侧向散射光的强度获得的散点图就能够准确获得血小板的光学信息，从而实现溶血条件下对血小板的准确计数。
根据另一种实施方式，所述方法进一步包括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。
在该实施方式中，使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信息进一步包括荧光信息，并且根据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。
在该实施方式中，根据一种方案，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第一荧光染料。
优选地，所述膜特异性染料选自DiA、DiD、DiI、DiO、DiR、DiS、FDA、Alexa Fluor488、Super Fluor 488及以它们为母体的变形结构所组成的组，以及所述线粒体特异性染料选自Janus Green B、MitoLite Red、罗丹明123和Mitotracker系列及以它们的母体的变形结构所组成的组。
在更优选的实施方式中，所述线粒体特异性染料为罗丹明123、Mitotracker Deep Red或Mitotracker Red。
在本发明中Mitotracker系列染料可包括Mitotracker Green、Mitotracker Deep Red和Mitotracker Red等。
在该方案中，根据荧光强度和前向散射光强度信息识别血小板。
进一步地，当根据前向散射光强度和荧光强度构成的散点图的预设区域中的粒子数超过预定阈值时报警网织红细胞。
在该实施方式中，根据又一种方案，所述荧光染料包括选自核酸特异性染料中的一种的第二荧光染料。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的染料。
在该方案中，所述方法进一步包括根据荧光强度和散射光强度信息识别网织红细胞。
更进一步地，所述方法还包括根据荧光强度和前向散射光强度信息进行网织红细胞计数。
在该实施方式中，根据再一种方案，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的第一荧光染料和选自核酸特异性染料中的一种的第二荧光染料。
在该方案中，所述方法进一步包括：根据荧光强度和侧向散射光强度信息区分血小板和网织红细胞。
本发明前述的血液检测方法，均能够根据所获得血小板的光学信息对血小板计数。
根据本发明的再一种实施方式，本发明的方法进一步在能够消除反射光对激光器的干扰的光学检测系统中进行检测，其中所述光学检测系统，包括：光学子系统、流动室、第一检测器；
所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，
所述激光器，配置为发射激光光束；
所述前光组件，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一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光为所述激光光束经所述流动室所产生。
根据具体实施方式，所述光隔离器由采用粘合方式相互连接的分光棱镜及偏振态转换组件构成；
所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
所述偏振态转换组件，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射。
根据另一具体实施方式，所述光隔离器由采用粘合方式相互连接的检偏器及偏振态转换组件组成；
所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
所述偏振态转换组件，配置为改变经所述检偏器的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光的反射光偏振态为S偏振光而被所述检偏器隔离。
在一种实施方式中，所述前光组件还包括准直透镜；所述准直透镜，沿所述激光光束的传播方向设置于所述激光器与所述光隔离器之间，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。
在一种实施方式中，所述后光组件还包括挡直光阑；所述前光组件，还配置为对所述激光光束进行前光处理，使得经所述前光处理的激光光束在第二方向上汇聚于所述挡直光阑处。
在一种实施方式中，所述前光组件还包括第一光汇聚组件及第二光汇聚组件；所述第一光汇聚组件，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所述流动室的血细胞被测样本处并产生散射光；所述第二光汇聚组件，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所述后光组件包括的挡直光阑处。
在一种实施方式中，所述后光组件还包括第三汇聚组件及小孔光阑；所述第三汇聚组件，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，并经所述小孔光阑的小孔进入所述第一检测器。
上述方案中，所述光学检测系统还包括第二检测器及荧光检测器；
所述第二检测器，配置为对与所述激光光束的传播方向所呈角度处于设定的角度范围内(如60°至120°)的散射光进行光强检测；
所述荧光检测器，配置为对所述血细胞被测样本所产生的荧光进行荧光检测。
上述方案中，所述第二方向与所述血细胞被测样本的流动方向垂直；
所述第一方向与所述血细胞被测样本的流动方向相同。
上述方案中，所述光隔离器的光隔离度不小于30db。
上述方案中，所述挡直光阑的光收集角度为1～10°。
上述方案中，所述激光光束的波长为630nm～640nm。
上述方案中，所述激光光束为P线偏振光。上述光学检测系统能够很好的隔离激光光束在光路中传播时产生的反射光，使得激光器能够稳定输出激光光束，进而避免反射光进入激光器产生的功率尖峰出现的小脉冲，也避免了这种干扰小脉冲和血小板粒子形成的小脉冲相互混淆的情况，更进一步提高了本发明方法对血小板的检测精度。
本发明在此还提供了第二种血液检测方法，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，所述溶血剂裂解所述血液样本中的红细胞；
使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信息；和
根据所述光学信息中的至少两种光学信息获得血小板的光学信息。
根据第一实施方式，所述溶血剂将所述血液样本中的红细胞裂解为其光散射特性显著不同于血小板的碎片。
在该第一实施方式中，所述第一试剂包括选自烷基糖苷、三萜皂苷、甾族皂苷中的至少一种溶血剂。
根据该第一实施方式，所述烷基糖苷选自具有通式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。
所述单糖可选自五碳糖、甲基五碳糖和六碳糖，其中五碳糖诸如阿拉伯糖、木糖、 核糖、来苏糖等；甲基五碳糖诸如夫糖、鼠李糖、鸡那糖等；六碳糖诸如葡萄糖、甘露糖、果糖、半乳糖、山梨糖；所述去氧单糖，诸如去氧核糖、去氧葡萄糖等；所述多糖诸如麦芽糖、蔗糖等。
n优选为6～14的整数，更优选为7～11的整数。
根据一种优选的实施方式，所述第一试剂进一步包括具有通式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 PCTCN2019084660-appb-000002
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。
所述通式II的非离子型表面活性剂中，优选地，R 1为C8-C18的直链烷基。C8-C18的直链烷基具体可为辛基、葵基、月桂基、十四烷基、十六烷基或硬脂基。更优选R 1为C12-C16的直链烷基，具体可为月桂基、十四烷基或十六烷基。R 2优选为-O-。m优选为15～30。
该实施方式中，所述第一试剂优选含有0.025g/L～10g/L、优选0.1g/L～5.0g/L的具有通式I的糖苷类化合物，和0.03～1.5g/L、优选0.05～1.0g/L的具有通式II的非离子型表面活性剂。
所述第一试剂还可包括选自缓冲剂、金属螯合剂、渗透压调节剂和防腐剂中的一种或多种。
在一种具体的实施方式中，所述血液样本与所述第一试剂的体积混合比可为1:40～1:60。用所述第一试剂在诸如40～60℃的温度下，反应15～100秒，优选反应40～80秒来处理所述血液样本。
根据该第一实施方式的血液检测方法，其中所述至少两种光学信息是前向散射光强度和侧向散射光强度。
根据该实施方式，所述试样中的红细胞经第一试剂处理后被深度裂解，而血小板仍保持其细胞形态，这样所得红细胞碎片的散射光性质与血小板的散射光性质出现了显著差异。由前向散射光和侧向散射光的强度获得的散点图就能够准确获得血小板的光学信息，从而实现溶血条件下对血小板的准确计数。
在该实施方式中，根据一种优选方案，所述方法进一步包括：在用所述第一试剂处理所述血液样本后，进一步用第二试剂进行处理，以获得待测样本。
其中所述第二试剂包含选自膜特异性染料和线粒体特异性染料中的一种。
在一个优选的实施方式中，所述膜特异性染料选自DiA、DiD、DiI、DiO、DiR、DiS、FDA、Alexa Fluor 488和Super Fluor 488。
根据另一种优选的实施方式，所述线粒体特异性染料选自Janus Green B、MitoLite Red、罗丹明123和Mitotracker系列。
在更优选的实施方式中，所述线粒体特异性染料为罗丹明123、Mitotracker Deep Red或Mitotracker Red。
在本发明中Mitotracker系列染料可包括Mitotracker Green、Mitotracker Deep Red和Mitotracker Red等。
在某些实施方式中，本发明的第二试剂进一步包括以上述染料为母体的变形结构。
当血液样本经含溶血剂的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。
因此，在该方案中，可根据荧光强度和侧向散射光强度信息识别血小板；根据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/或当根据前向散射光强度和荧光强度构成的散点图的预设区域中的粒子数超过预定阈值时报警网织红细胞。
在该实施方式中，根据另一种优选方案，所述第二试剂包含一种核酸特异性染料，或者同时包含一种选自膜特异性染料和线粒体特异性染料中的染料以及一种核酸特异性染料。该方案中，红细胞裂解后散落的细胞器，经核酸特异性染料染色，可进一步使具有细胞核的网织红细胞的碎片与血小板区分，并同时可以获得网织红细胞的准确光学信息，从而利用该第一实施方式的方法，可在一次光学检测中同时获得血小板、白细胞和网织红细胞的光学信息。
因此，根据该优选方案，所述血液检测方法进一步包括：根据荧光强度和散射光强度信息区分血小板和网织红细胞。并更优选地，根据荧光强度和前向散射光强度信息区分血小板和网织红细胞，并能对网织红细胞进行计数。在该第二种血液检测方法中的第一实施方式中的全部内容可同样适用于本发明的第一种血液检测方法。同样的，前述第一种血液检测方法中的全部内容也适用于第二种血液检测方法中的第一实施方式。
根据本发明的第二实施方式，所述血液检测方法包括：用第一试剂处理所述血液样本，并用第二试剂进行处理，以获得待测样本，其中所述第一试剂包括溶血剂。
根据该第二实施方式，所述溶血剂没有特别限制，本领域常规的红细胞溶血剂都可用于该实施方式中。
该实施方式的所述第二试剂与上述第一实施方式相同，可包含选自上述膜特异性染料和上述线粒体特异性染料中的一种。
同样的，当血液样本经含溶血剂的第一试剂和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使血小板区分于被裂解的红细胞碎片。由于膜特异性染料或线粒体特异性染料的使用，在该实施方式中，红细胞仅经常规溶血剂裂解就可通过二维散点图与血小板明显区分，而不必如第一实施方式中那样对红细胞进行深度裂解。
在该实施方式中，可根据荧光强度和侧向散射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；根据荧光强度和前向散射光强度信息全区分血小板；和/或当根据前向散射光强度和荧光强度构成的散点图的预设区域中的粒子数超过预定阈值时报警网织红细胞。
同样地，该实施方式中，所述第二试剂可进一步包含选自核酸特异性染料中的一种荧光染料。并同样地由此可进一步根据荧光强度和前向散射光强度信息区分血小板和网织红细胞，优选地进一步对网织红细胞计数。
根据第三实施方式，上述本发明的血液检测方法，可通过光学检测系统的检测获得所述光学信息，所述光学检测系统包括：
包括：光学子系统、流动室、第一检测器；
所述光学子系统包括：激光器、包括光隔离器的前光组件及包括挡直光阑的后光组件；其中，
所述激光器，配置为发射激光光束；
所述前光组件，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所述挡直光阑处，在第一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述 散射光及汇聚于所述挡直光阑处的激光光束进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
所述光隔离器，配置为隔离所述激光光束经所述流动室及所述后光组件所产生的反射光。
上述方案中，所述光隔离器由采用粘合方式相互连接的分光棱镜及偏振态转换组件构成；
所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
所述偏振态转换组件，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射。
上述方案中，所述光隔离器由采用粘合方式相互连接的检偏器及偏振态转换组件组成；
所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
所述偏振态转换组件，配置为改变经所述检偏器的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光的反射光偏振态为S偏振光而被所述检偏器隔离。
上述方案中，当所述激光光束垂直入射所述光隔离器时，所述分光棱镜的第一入射面的反射率不大于0.5％。
上述方案中，所述光隔离器由采用粘合方式相互连接的带通滤光片及倍频晶体组成；
所述带通滤光片，配置为使波长为λ的所述激光光束通过；
所述倍频晶体，配置为对经所述带通滤光片的激光光束进行倍频，并对所述倍频后的激光光束的反射光再次进行倍频，而被所述带通滤光片滤除。
上述方案中，所述前光组件还包括准直透镜；
所述准直透镜，沿所述激光光束的传播方向设置于所述激光器与所述光隔离器之间，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。
上述方案中，所述前光组件还包括第一光汇聚组件及第二光汇聚组件；
所述第一光汇聚组件，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所述流动室的血细胞被测样本处并产生散射光；
所述第二光汇聚组件，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所述挡直光阑处。
上述方案中，所述后光组件还包括第三汇聚组件及小孔光阑；
所述第三汇聚组件，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，并经所述小孔光阑的小孔进入所述第一检测器。
上述方案中，所述光学检测系统还包括第二检测器及荧光检测器；
所述第二检测器，配置为对与所述激光光束的传播方向所呈角度处于设定的角度范围内(如60°至120°)的散射光进行光强检测；
所述荧光检测器，配置为对所述血细胞被测样本所产生的荧光进行荧光检测。
上述方案中，所述第二方向与所述血细胞被测样本的流动方向垂直；
所述第一方向与所述血细胞被测样本的流动方向相同。
上述方案中，所述光隔离器的光隔离度不小于30db。
上述方案中，所述挡直光阑的光收集角度为1～10°。
上述方案中，所述激光光束的波长为630nm～640nm。
上述方案中，所述激光光束为P线偏振光。上述光学检测系统能够很好的隔离激光光束在光路中传播时产生的反射光，使得激光器能够稳定输出激光光束，进而避免反射 光进入激光器产生的功率尖峰出现的小脉冲，也避免了这种干扰小脉冲和血小板粒子形成的小脉冲相互混淆的情况，更进一步提高了本发明方法对血小板的检测精度。
根据本发明另一方面的目的，本发明还提供一种血液分析系统，包括：
采样部，用于获取血液样本，并将所述血液样本输送到所述反应部；
试剂供应部，用于贮存至少第一试剂并根据需要供应到所述反应部；
反应部，包括混合室，用于将所述血液样本与第一试剂混合以形成待测试样，其中所述第一试剂包括溶血剂，所述溶血剂裂解所述血液样本中的红细胞；
光学检测系统，包括流动室和至少第一检测器，用于当所述待测试样由所述混合室被输送到所述光学系统并使所述待测试样中的粒子逐个通过流动室到达检测区时，所述第一检测器对待测试样中的粒子进行检测以获得所述待测试样中的光学信息；和
数据处理模块，其与所述光学系统可操作地连接，并包括处理器和存储有计算机程序的非暂时性计算机可读存储介质，其中当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述光学信息中的至少两种光学信息获得血小板的光学信息。
根据本发明血液分析系统的第一实施方式，所述溶血剂将所述血液样本中的红细胞裂解为其光散射特性显著不同于血小板的碎片，且其中所述处理模块中，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：
根据所获得的光学信息中的前向散射光强度和侧向散射光强度对血小板计数；和/或
根据前向散射光强度和侧向散射光强度将白细胞区分为至少包括单核细胞、淋巴细胞和中性粒细胞的亚群。
根据本发明血液分析系统的第二实施方式，其中，
所述混合室用于所述血液样本与第二试剂混合以形成待测试样，其中所述第二试剂包含选自膜特异性染料和线粒体特异性染料中的一种荧光染料；
所述光学检测系统包括第二检测器，所述第二检测器为荧光检测器，以便当所述待测试样中的粒子逐个通过检测区时进一步获得荧光信号；和
所述数据处理模块中，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：
根据荧光强度和侧向散射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；
根据荧光强度和前向散射光强度信息全区分血小板；和/或
当根据前向散射光强度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报警网织红细胞。
在该第二实施方式中，根据另一方案，所述第二试剂进一步包含选自核酸特异性染料中的一种荧光染料，和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：根据荧光强度和前向散射光强度信息区分血小板和网织红细胞，优选地进一步对网织红细胞计数。
根据本发明血液分析系统的第三实施方式，其中所述光学检测系统，进一步包括：光学子系统，
所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，
所述激光器，配置为发射激光光束；
所述前光组件，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一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强 检测；
所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光至少为所述激光光束经所述流动室所产生。
应理解，前述本发明的光学检测系统的全部特征可应用于在本发明的血液分析系统的光学检测系统中。
本发明提供了一种全新的血液检测方法，该方法对血液样本进行溶血处理，并通过光学检测能够实现对血小板的准确检测，并且该方法能够同时获得白细胞的分析结果，并在使用核酸特异性染料的情况下进一步获得网织红细胞的检测信息，从而首次实现了溶血条件下利用光学信息区分血小板，并且在单一通道中能够同时获得白细胞，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为本发明实施例提供的光学检测系统的组成结构示意图一；
图2为本发明实施例提供的光学检测系统的组成结构示意图二；
图3为本发明实施例提供的光隔离器的原理示意图；
图4为本发明实施例提供的光隔离器的第一入射面的示意图；
图5为本发明实施例提供的第二柱面镜的光汇聚原理示意图；
图6为本发明实施例提供的挡直光阑的正面视图；
图7为本发明实施例提供的血细胞分析仪的组成结构示意图；
图8为本发明实施例提供的光学检测系统的组成结构示意图三；
图9为本发明实施例提供的光学检测系统的组成结构示意图四；
图10为实施例1中获得的经深度溶血的试样在流式细胞仪上收集血小板(A)与白细胞(B)的散点图；
图11为实施例2经深度溶血的试样(A)和经常规溶血剂溶血后的试样(B)PLT展出对比；
图12为实施例3在深度溶血状态下对血小板和网织红细胞检测得到的荧光-前向散射光的光强度散点图(A)，和白细胞的荧光-侧向散射光的光强度散点图(B)；
图13为实施例4在深度溶血状态下对血小板和网织红细胞检测得到的荧光-前向散射光的光强度散点图(A)，和白细胞的荧光-侧向散射光的光强度散点图(B)；
图14为实施例5在深度溶血状态下加入核酸染料对血小板和网织红细胞检测得到的荧光-前向散射光的光强度散点图(A)，和白细胞的荧光-侧向散射光的光强度散点图(B)；
图15为实施例6在深度溶血状态下加入膜染料对血小板和网织红细胞检测得到的荧光-前向散射光的光强度散点图(A)，和白细胞的荧光-侧向散射光的光强度散点图(B)；
图16为实施例7在深度溶血状态下加入线粒体染料对血小板和网织红细胞检测得到的荧光-侧向散射光的光强度散点图；
图17为实施例8在深度溶血状态下加入膜染料和核酸染料对血小板和网织红细胞检测得到的荧光-前向散射光的光强度散点图(A)，和白细胞的荧光-侧向散射光的光强度散点图(B)；
图18为实施例9测试血小板(A)及网织红细胞(B)相关的图；
图19示出了实施例10溶血状态下，染料Alexa Fluor 488对(A)血小板和(B)白细胞的染色及区分效果；
图20示出了实施例11溶血状态下，染料DiD对(A)血小板和(B)白细胞的染色及区分效果；
图21示出了实施例12溶血状态下，染料罗丹明123对(A)血小板和(B)白细胞的染色及区分效果；
图22示出了实施例13溶血状态下，染料Mitotracker Deep Red对(A)血小板和(B)白细胞的染色及区分效果；
图23示出了实施例14溶血状态下，染料Mitotracker Red对(A)血小板和(B)白细胞的染色及区分效果；
图24为实施例15的染色法与迈瑞6800仪器上在测试值上的相关性。
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，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。这些方法都是在不溶血的前提下，在单独的检测通道中进行。
通常，红细胞溶血剂在破坏红细胞膜的同时也会对其他细胞膜造成损伤。而且，溶血后产生的大量红细胞碎片，通常会认为将对血小板的检测造成更大的干扰。因此，目前已知的光学检测血小板的方法中大多采用不溶血的方法。
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。
本发明人开发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区分开，并且，还能同时获得白细胞的光学信息，并且在采用核酸特异性染料的情况下进一步获得网织红细胞的光学信息。
以下对本发明的第二种血液检测方法及相应的血液分析系统示例性地详细说明本 发明。
根据本发明的血液检测方法，首先用包括溶血剂的第一试剂处理血液样本以获得待测试样，然后通过光学检测设备对所述试样进行检测，并利用至少两种光学信息将血小板与试样中的其他粒子区分开，从而获得血小板的光学信息。经验证，本发明的方法获得的血小板计数与现有的血液分析仪以其他方法检测得到的结果具有高度的一致性。
更进一步地，本发明的方法在常规的白细胞计数通道中，对白细胞的计数不产生影响。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，甚至在利用荧光染料的情况下可获得四分类的白细胞检测结果，并可对网织红细胞报警，或者计数。
以下通过具体实施方式来进一步说明本发明的方法。
根据本发明的第一实施方式，所述第一试剂中包含能够使红细胞深度裂解的溶血剂。所述溶血剂没有特别限制，可采用本发明中红细胞深度溶血剂。举例来说，这样的溶血剂可为烷基糖苷、三萜皂苷、甾族皂苷等等。
一种具体的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。
通式(I)中，单糖没有特别限制，常用的可选自五碳糖、甲基五碳糖和六碳糖，但不限于此。五碳糖诸如阿拉伯糖、木糖、核糖、来苏糖等。甲基五碳糖诸如夫糖、鼠李糖、鸡那糖等。六碳糖诸如葡萄糖、甘露糖、果糖、半乳糖、山梨糖。去氧单糖也没有特别限制，诸如去氧核糖、去氧葡萄糖等，但不限于此。多糖诸如麦芽糖、蔗糖等，但不限于此。n优选为6～14的整数，更优选为7～11的整数。
所述通式I的糖苷类化合物具体可为辛基葡萄糖苷，壬基葡萄糖苷，葵基葡萄糖苷，十二烷基麦芽糖苷，十四烷基麦芽糖苷，十二烷基葡萄糖苷，优选辛基葡萄糖苷，壬基葡萄糖苷，葵基葡萄糖苷，十二烷基麦芽糖苷，更优选葵基葡萄糖苷，十二烷基麦芽糖苷。
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。
该第一实施方式中的第一试剂，优选进一步包括具有通式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 PCTCN2019084660-appb-000003
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。
根据优选的实施方式，所述通式II的非离子型表面活性剂中，R 1为C8-C18的直链烷基。C8-C18的直链烷基具体可为辛基、葵基、月桂基、十四烷基、十六烷基或硬脂基。更优选R 1为C12-C16的直链烷基，具体可为月桂基、十四烷基或十六烷基。R 2优 选为-O-。m为10～50，优选为15～30。
所述通式II的非离子型表面活性剂具体实例可为十六烷醇聚氧乙烯(15)醚、十二烷醇聚氧乙烯(21)醚、十六烷醇聚氧乙烯(23)醚、十六烷醇聚氧乙烯(25)醚、十六烷醇聚氧乙烯(30)醚，但不限于此。
通式II的非离子型表面活性剂的浓度没有特别限制，但可为0.03～1.5g/L，优选0.05～1.0g/L。
在本发明中，该非离子表面活性剂可以以单一物质使用，也可以两者或更多种的混合物来使用。取决于所使用的非离子表面活性剂的种类，其在溶血剂中的浓度也是不同的。通常来说，烷基链越长、聚氧乙烯部分的重复单元数目越多的非离子型表面活性剂，其浓度相对较低。
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。所述有机酸优选为未取代的或被羟基或氨基取代的C1-6烷基的一元、二元或三元羧酸；未取代的或被羟基或氨基取代的C1-6烷基的磺酸；C6-10芳基C1-6烷基酸；C6-10芳基二(C1-6烷基酸)；和C6-10芳基磺酸所组成的组中。
所述有机酸及其盐的具体实例可为甲酸、乙酸、苯甲酸、柠檬酸(3-羟基-1,3,5-戊三酸)、苹果酸(2－羟基丁二酸)、苯二甲酸、苯磺酸、α-萘磺酸、牛磺酸等及它们的碱金属盐，诸如钠盐、钾盐，但不限于此。
所述有机酸或有机酸盐在溶血剂中的浓度为0.05g/L到2g/L，优选0.1g/L到0.5g/L。
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。缓冲剂，例如可以为选自磷酸及其盐、柠檬酸及其盐、TRIS等中一种，通常为两种或更多种构成的缓冲体系。金属螯合剂，用作抗凝剂，常见的如EDTA钠。渗透压调节剂，通常为无机盐类，诸如氯化钠、硫酸钠、硫酸钾，硼酸钠等。防腐剂，例如异噻唑啉酮、叠氮钠，咪唑烷基脲。
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，可以获得对于红细胞程度较深的裂解。但是由于任何溶血剂会对任何细胞膜产生作用，因此，本文中红细胞深度裂解(深度溶血)指反应条件的选择使红细胞相比常规被进一步地裂解，但血小板可基本保持其细胞形态，优选也使白细胞能基本保持其细胞形态。在光信号的散点图上，血小板和被裂解的红细胞能形成区分开的两个群。于此相对，本文中的红细胞常规裂解指使用常规的溶血剂，反应后，被裂解的红细胞会混在血小板粒子群的情况。
通过以下具体实施例将详述的，第一实施方式的方法，能够对红细胞造成更深程度的裂解，使红细胞的细胞膜破碎成更小的碎片，从而通过光学检测得到的前向和侧向散射光强度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检测、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。
根据一种优选方案，经第一试剂处理的血液样本还使用第二试剂进行处理。
该第二试剂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 Fluor488、Super Fluor 488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。
本发明的第一实施方式中的第一试剂包含溶血能力强的表面活性剂，使得红细胞的碎片体积更小。溶血后从网织红细胞(RET)中散出的细胞器颗粒，其数目与RET的值有一定的相关性。由于网织红细胞是有核红细胞，因而加入核酸特异性荧光染料后，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(RET)的血液样本，单纯用前散射光和侧散射光信号检测时，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粒子进行计数，当计数值超过预定值(例如超过血小板数量达到一定程度)时，可进行网织红细胞报警，以便进一步对受试者进行检查。
采用该优选的方案，可获得上述三分类甚至四分类的白细胞亚群，并可识别幼稚粒细胞。
根据优选的方案，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。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中的一种，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。
以上详述的本发明第二种血液检测方法中的第一实施方式可全部应用于本发明第一种血液检测方法中。因此根据本发明的第一种血液检测方法就不再赘述。
本发明的第二实施方式，用第一试剂和第二试剂对血液样本进行处理来获得待测试样。
在该实施方式中，第一试剂包含任意的溶血剂，只要能裂解红细胞即可，对其溶血程度没有特别限制，可以是常规的溶血剂。示例性的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。
第二试剂包含选自上述膜特异性染料和上述线粒体特异性染料中的一种。
该实施方式中，当血液样本经含溶血剂的第一试剂和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的荧光特性显著不同，因而可通过检测荧光强度和一种散射光强度，特别是通过检测荧光强度和前向散射光强度可使血小板很好地区分于被裂解的红细胞碎片。由于膜特异性染料或线粒体特异性染料的使用，在该实施方式中，红细胞仅经溶血剂裂解就可通过二维散点图与血小板明显区分，而不必如第一实施方式中那样对红细胞进行深度裂解。而且该方案可同样通过荧光强度和侧向散射光强度信息获得白细胞的亚群和计数，以及当存在网织红细胞时进行报警。
该实施方式中，所述第二试剂可进一步包含一种核酸特异性染料。并同样地，由此可进一步获得网织红细胞的光学信息，并优选地计数。
在第三实施方式中，本发明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系统中进行检测，以获取更为准确的血小板计数。
常规的光学检测系统中的激光器易受到光路中反射光的影响而不稳定，产生振幅变化、频率移动或功率尖峰等。在实际应用中，当激光器不稳定振荡产生功率尖峰时，其在光学前向信号上的表现是小脉冲。这些干扰小脉冲和血小板粒子在检测中形成的小脉冲相互混淆，造成干扰。
参考图1～9，对本发明的光学检测系统进行详细说明。
发明人在研究过程中发现，为阻止反射光回馈到激光器中，可以采用基于法拉第效应的磁光器件作为光隔离器，放置于光路中。这种隔离器机械尺寸较大，一般只能放置在光源组件外部，而光源组件外部的光束为非平行光，当非平行光入射光隔离器时，其光隔离的效果会受到严重削弱。因此这种方案存在尺寸大、成本高、隔离效果不好的缺点。
在本发明实施例中，光学检测系统包括：光学子系统、流动室、第一检测器；所述光学子系统包括：激光器、包括光隔离器的前光组件及包括挡直光阑的后光组件；其中，
所述激光器，配置为发射激光光束；
所述前光组件，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所述挡直光阑处，在第一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光及汇聚于所述挡直光阑处的激光光束进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
所述光隔离器，配置为隔离所述激光光束经所述流动室及所述后光组件所产生的反射光。
接下来对本发明实施例提供的光学检测系统进行详细说明。
图1为本发明实施例提供的光学检测系统的组成结构示意图一，图2为本发明实施例提供的光学检测系统200的组成结构示意图二，结合图1、图2所示，本发明实施例提供的光学检测系统200包括：光学子系统1、流动室2、第一检测器3；
所述光学子系统1包括：激光器11、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，
所述激光器11，配置为发射激光光束；
所述前光组件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所述挡直光阑131处，在第一方向上汇聚于所述流动室2的血细胞被测样本处并产生散射光；
所述后光组件13，沿所述激光光束的传播方向设置于所述流动室2之后，配置为对所述散射光及汇聚于所述挡直光阑131处的激光光束进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器3进行光强检测；
所述光隔离器121，配置为隔离所述激光光束经所述流动室及所述后光组件所产生的反射光。
接下来对光学子系统中的激光器11进行说明，在一实施例中，所述激光器11为半导体激光器，实际实施时，可以为P线偏振激光器；在实际应用中，激光器发射激光光束的波长决定了光路主要参数的设计，比如透镜的选型、信号收集角度的选择等，且激光光束的波长，也和检测中用到的试剂如荧光染料有关，在一实施例中，激光器11发出的激光光束的波长为630nm～640nm。
接下来对光学子系统中的前光组件12中的各部分进行说明。在一实施例中，前光组件12中的光隔离器121由采用粘合方式相互连接的分光棱镜及偏振态转换组件构成；
所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
所述偏振态转换组件，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射。
以偏振态转换组件为1/4波片为例，参见图3，图3为本发明实施例提供的光隔离器的原理示意图，当激光光束L(TM模)垂直入射分光棱镜35时，激光光束L中的P偏振光31(即平行纸面的偏振分量)能够通过分光棱镜35，而激光光束L中的S偏振光32(即垂直纸面的偏振分量)被分光棱镜35的45°斜面反射出去；继而，P偏振光31透过1/4波片36，经过1/4波片36后的P偏振光31的偏振态发生改变，由线偏振光变成圆偏振光33；圆偏振光33的反射光(被后级光路37反射形成的反射光)再次经过1/4波片36后，偏振态发生改变，由圆偏振光转换成S偏振光34，而S偏振光34会被分光棱镜35的45°斜面反射出去，不会回馈到激光器，从而实现对光路反射光的隔离。
这里，在实际应用中，当激光光束垂直入射光隔离器时，分光棱镜的第一入射面的反射率不大于0.5％，或者不大于0.1％，或者不大于0.05％。这里的第一入射面参见图 4，图4为本发明实施例提供的光隔离器的第一入射面的示意图，其中，标号41所示即为第一入射面，在实际实施时，第一入射面的光反射率可由第一入射面的镀膜设计与工艺实现。
在一实施例中，光隔离器121还可由采用粘合方式相互连接的检偏器及偏振态转换组件组成；
所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
所述偏振态转换组件，配置为改变经所述检偏器的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的反射光的偏振态为S偏振光而被所述检偏器隔离。
以偏振态转换组件为磁光晶体为例进行说明，当激光光束入射检偏器时，仅激光光束中的P偏振光能够通过检偏器，经过检偏器的P偏振光进入磁光晶体，经过磁光晶体的P偏振光的偏振态发生改变，其偏振方向旋转45°。该偏振光被后级光路反射形成的反射光再次经过磁光晶体后，偏振方向再次旋转45°，形成与原P偏振光的偏振态垂直的S偏振光而被检偏器隔离，不会回馈到激光器。
在实际应用中，所述偏振态转换组件还可以为旋光晶体，在实际应用中，分光棱镜、检偏器可以和1/4波片、磁光晶体、旋光晶体中任一个进行组合实现光路中反射光的隔离。
在一实施例中，所述光隔离器121由采用粘合方式相互连接的带通滤光片及倍频晶体组成；
所述带通滤光片，配置为使波长为λ的所述激光光束通过；
所述倍频晶体，配置为对经所述带通滤光片的激光光束进行倍频，并对所述倍频后的激光光束的反射光再次进行倍频，而被所述带通滤光片滤除。
在一实施例中，所述光隔离器的光隔离度不小于30db。
在一实施例中，所述前光组件12还包括准直透镜122；
所述准直透镜122，沿所述激光光束的传播方向(光轴方向)设置于所述激光器11与所述光隔离器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；
所述第一光汇聚组件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所述流动室的血细胞被测样本处并产生散射光；
所述第二光汇聚组件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所述档直光阑131处。
这里，在实际应用中，所述第二方向为横向，即与所述血细胞被测样本的流动方向垂直的方向；所述第一方向为纵向，即与所述血细胞被测样本的流动方向相同的方向。在实际应用中，第一光汇聚组件123和第二光汇聚组件124可以为光学参数(如焦距等)不同的柱面镜实现，如第一光汇聚组件123由第一柱面镜实现，第二光汇聚组件124由第二柱面镜实现。
以第二光汇聚组件124为第二柱面镜为例进行说明，参见图5，图5为本发明实施例提供的第二柱面镜的光汇聚原理示意图，激光光束经过第二柱面镜，第二柱面镜在纵向上不对激光光束进行处理，经第二柱面镜的激光光束在横向上被压缩，激光光束在横向(垂直于血细胞被测样本的流动方向)上聚焦，在本发明实施例中聚焦于档直光阑处。
接下来对后光组件13中各部分进行说明，参见图6，图6为本发明实施例提供的挡直光阑的正面视图，照射到流动室中血细胞样本处的激光光束发生散射，产生的散射光被挡直光阑所收集，在实际实施时，挡直光阑对低角度散射信号的收集角度进行约束；同时，挡直光阑在第二方向上汇聚于挡直光阑处的激光光束进行阻挡。在一实施例中， 挡直光阑的光收集角度可以为1～10°。
在一实施例中，所述后光组件13还包括第三汇聚组件132及小孔光阑133；
所述第三汇聚组件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，并经所述小孔光阑的小孔进入所述第一检测器，以进行光强检测。
在一实施例中，第三汇聚组件可以为以下之一：
至少一个平凸透镜与至少一个双凸透镜构成的透镜组；
至少两个平凸透镜构成的透镜组；
至少两个双凸透镜构成的透镜组；
至少两个球面透镜构成的透镜组；
非球面镜。
在一实施例中，所述光学检测系统还包括第二检测器4及荧光检测器5；
所述第二检测器，沿与所述激光光束的传播方向所呈角度处于设定的角度范围内的方向设置，配置为对与所述激光光束的传播方向所呈角度处于设定的角度范围内的散射光进行光强检测；
所述荧光检测器，沿与所述激光光束的传播方向所呈角度处于设定的角度范围内的方向设置，配置为对所述血细胞被测样本所产生的荧光进行荧光检测。
接下来对本发明实施例提供的血液分析系统进行说明，图7为本发明实施例提供的血液分析系统的组成结构示意图。根据本发明的血液分析系统大体上包括采样部，试剂供应部、反应部、光学检测系统和数据处理模块。
图7示出了根据本发明的一种具体的血液分析系统。该血液分析系统具体第一机壳100、第二机壳200、采样部10、试剂供应部(未示出)、反应部30、光学检测系统50、数据处理模块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，光学检测系统50及数据处理模块70设置在第二机壳200的内部，分别设置在第二机壳200两侧。反应部30设置在第一机壳100的内部，输出部90、采样部10在第一机壳100的外表面。
所述采样部10，具有采样针，采集血液样本，并将采集的血液样本输送至反应部30。
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)并根据需要将相应的试剂供应到所述反应部。
所述反应部30，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，得到包含多个所述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系统的流动室。
所述光学检测系统50包括：光学子系统、流动室和第一检测器。所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，所述激光器，发射激光光束；所述前光组件，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一方向上汇聚于所述流动室的血液粒子(如血小板粒子)处并产生散射光；所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光为所述激光光束经所述流动室及所述后光组件所产生。所述流动室，供诸如血小板粒子的血液粒子排队通过。第一检测器用于检测经过流动室的血液粒子的光学信息，尤其是光强度信息。
所述数据处理模块70，配置为根据所述第一检测器检测得到的所述散射光的光强信号，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。
所述输出部90，配置为输出对应所述血液(例如血小板)粒子的检测结果。
接下来对血液分析系统包括的光学检测系统50所包括的各部分进行说明。
图8为本发明实施例提供的光学检测系统的组成结构示意图，如图8所示，本发明实施例提供的光学检测系统包括：激光器71、前光组件72、流动室73、后光组件74、前向检测器75、侧向检测器76及荧光检测器77。
在实际应用中，血液粒子(尤其以血小板粒子为例)从流动室穿过而被检测(如光强检测)、计数等，在本实施例中，血小板粒子的流动方向为垂直于纸面方向，激光器71发射的激光光束的传播方向为平行于纸面方向。
在实际实施时，激光器71为P线偏振激光器，激光器71发出的激光光束的波长为630nm—640nm。
参见图8，前光组件72包括沿所述激光光束的传播方向依次设置的准直透镜721、光隔离器722、第一柱面镜723及第二柱面镜724；后光组件74包括沿所述激光光束的传播方向依次设置的挡直光阑741、平凸透镜742、双凸透镜743及小孔光阑744。
激光器71发出的激光光束经准直透镜721的准直处理，形成平行激光光束，然后透过光隔离器722，经第一柱面镜723在纵向(垂直于纸面方向)上聚焦于流动室73中心处作为检测光斑，所述激光光束的传播方向与所述流动室的光入射面之间的夹角呈直角，聚焦于流动室的激光光束照射到流动室中的血小板粒子而产生光散射，经第一柱面镜723在纵向的压缩，激光光束在纵向上的尺寸与血小板粒子的尺寸相匹配，经第一柱面镜723的激光光束，进入第二柱面镜724，在横向(平行于纸面方向)上被压缩，在横向上汇聚于挡直光阑741处。
其中，照射到流动室中的血小板粒子产生的散射光经挡直光阑的收集，进而被平凸透镜742、双凸透镜743组成的透镜组聚焦，汇聚于小孔光阑744处，经小孔光阑744的小孔进入前向检测器75，而被前向检测器75进行前向光信号强度检测。
在实际应用中，如图8所示，侧向检测器76及荧光检测器77沿与所述激光光束的传播方向垂直的方向设置；其中，侧向检测器76配置为对与所述激光光束的传播方向垂直的散射光进行光强检测；荧光检测器77，沿与所述激光光束的传播方向垂直的方向设置，配置为对所述散射光进行荧光检测。
在实际实施时，由激光器71发出的激光光束在图8所示光路中传播会形成反射光，光隔离器722配置为隔离激光器71发出的激光光束在光路中传播所产生的反射光。
在实施例中，光隔离器722由采用粘合方式相互连接的分光棱镜及1/4波片构成；
所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
所述1/4波片，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射。
当激光光束垂直入射分光棱镜时，激光光束中的P偏振光(即平行纸面的偏振分量)能够通过分光棱镜，而激光光束中的S偏振光(即垂直纸面的偏振分量)被分光棱镜的45°斜面反射出去；继而，P偏振光透过1/4波片，经过1/4波片后的P偏振光的偏振态发生改变，由线偏振光变成圆偏振光；圆偏振光的反射光(被后级光路反射形成的反射光)再次经过1/4波片后，偏振态发生改变，由圆偏振光转换成S偏振光，而S偏振光会被分光棱镜的45°斜面反射出去，不会回馈到激光器，从而实现对光路反射光的隔离。
这里，在实际应用中，当激光光束垂直入射光隔离器时，分光棱镜的第一入射面的反射率不大于0.5％。这里的第一入射面参见图4，图4为本发明实施例提供的光隔离器的第一入射面的示意图，其中，标号41所示表面即为第一入射面，在实际实施时，第一入射面的光反射率可由第一入射面的镀膜设计与工艺实现。
在一实施例中，光隔离器121还可由采用粘合方式相互连接的检偏器及偏振态转换 组件组成；
所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
所述磁光晶体，配置为改变经所述检偏器的所述P偏振分量的偏振态，使其偏振方向旋转45°。该偏振光被后级光路反射形成的反射光再次经过磁光晶体后，偏振方向再次旋转45°，形成与原P偏振光的偏振态垂直的S偏振光，从而被所述检偏器隔离。
当激光光束入射检偏器时，仅激光光束中的P偏振光能够通过检偏器，经过检偏器的P偏振光进入磁光晶体，经过磁光晶体的P偏振光的偏振态发生改变，其偏振方向旋转45°。该偏振光被后级光路反射形成的反射光再次经过磁光晶体后，偏振方向再次旋转45°，形成与原P偏振光的偏振态垂直的S偏振光，从而被所述检偏器隔离，不会回馈到激光器。
在一实施例中，所述光隔离器还可由采用粘合方式相互连接的带通滤光片及倍频晶体组成；
所述带通滤光片，配置为使波长为λ的所述激光光束通过；
所述倍频晶体，配置为对经所述带通滤光片的激光光束进行倍频，并对所述倍频后的激光光束的反射光再次进行倍频，而被所述带通滤光片滤除。
在一实施例中，所述光隔离器的光隔离度不小于30db。
在一实施例中，所述激光光束的传播方向与所述流动室的光入射面之间的夹角可以为非直角，即对上述实施例中的流动室的位置在x-y平面即纸面进行一定角度的偏转，使得所述流动室倾斜放置，图9为本发明实施例提供的光学检测系统的组成结构示意图，如图9所示，激光光束的传播方向与所述流动室的光入射面之间的夹角θ为锐角，在一实施例中，θ角的范围为80°～90°，如此，当激光光束照射至流动室内部时，光束在流动室内部表面发生反射后，反射光偏离光轴(激光光束传播方向)，从而减少进入激光器的反射光的光通量。本领域技术人员可知，只要能使流动室光入射面的反射光不进入前光组件的θ角都可以适用本申请。通过深入研究发现，虽然流动室倾斜放置可能会使前向散射光的光路偏移，荧光和侧向散射光的收集角度发生变化，但进一步深入研究发现，可以通过调整后光组件在光路中的位置弥补前向散射光的影响，同时，通过大量的研究实验证明对荧光和侧向散射光的检测可以接受，不影响最终的检测结果。
应用本发明上述实施例，血小板粒子较小，流经流动室形成小脉冲，由于上述血细胞分析仪中的光学检测系统中光隔离器能够很好的隔离激光光束在光路中传播时产生的反射光，使得激光器能够稳定输出激光光束，避免了反射光进入激光器产生的功率尖峰出现的小脉冲，也即避免了干扰小脉冲和血小板粒子形成的小脉冲相互混淆，极大的提高了检测精度。
本发明实施例还提供了一种血小板检测方法，应用于上述包括光学检测系统的血细胞分析仪，所述光学检测系统包括：激光器、包括光隔离器的前光组件、后光组件、流动室及第一检测器；所述方法包括：
提供含有血小板的待测试液；
使待测试液中的血小板粒子逐个通过流动室的检测区；
使用所述前光组件对激光器发射的激光光束进行前光处理，使得经所述前光处理的激光光束在第一方向上汇聚于流动室的检测区，所述血小板粒子经过所述检测区产生散射光；
使用所述后光组件对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入第一检测器；
其中，所述激光光束经所述流动室及所述后光组件所产生的反射光被所述光隔离器所隔离；
使用所述第一检测器对进入的所述散射光进行光强检测，得到第一检测结果，以基 于所述第一检测结果识别所述血小板粒子。
在一实施例中，还包括：
对采集的血液样本进行溶血处理，使得所述血液样本中的红细胞被裂解，得到包含多个所述血小板粒子的待测试液。
在一实施例中，还包括：
使用前光组件对所述激光光束进行前光处理，使得经所述前光处理的激光光束在第二方向上汇聚于所述后光组件包括的挡直光阑处。
在一实施例中，所述血细胞分析仪还包括第二检测器；相应的，所述方法还包括：
使用所述第二检测器对与所述激光光束的传播方向所呈角度处于设定的角度范围内的散射光进行光强检测，得到第二检测结果，以结合所述第一检测结果及所述第二检测结果识别所述血小板粒子。
在一实施例中，所述血细胞分析仪还包括荧光检测器；相应的，所述方法还包括：
所述待测试液中的血小板粒子经特定荧光染料染色处理；所述特定荧光染料可以为膜染料、线粒体染料中至少一种；
所述待测试样中的血小板粒子经过所述检测区时，还产生荧光，所述荧光检测器，配置为对所述血小板粒子所产生的荧光进行检测，得到第三检测结果，以结合所述第一检测结果及所述第三检测结果识别所述血小板粒子。
在一实施例中，所述血细胞分析仪还包括第二检测器及荧光检测器；相应的，所述方法还包括：
所述待测试液中的血小板粒子经荧光染色处理；
所述待测试液中的血小板粒子经过所述检测区时，还产生荧光，
所述第二检测器，配置为对与所述激光光束的传播方向所呈角度处于设定的角度范围内的散射光进行光强检测，得到第二检测结果；其中，所述设定的角度范围可以为60°120°。
所述荧光检测器，配置为对所述血小板粒子所产生的荧光进行检测，得到第三检测结果；
结合所述第一检测结果、所述第二检测结果及所述第三检测结果识别所述血小板粒子和白细胞计数和分类。
应用本发明上述实施例，能够很好的隔离激光光束在光路中传播时产生的反射光，使得激光器能够稳定输出激光光束，进而避免反射光进入激光器产生的功率尖峰出现的小脉冲，也避免了当血细胞被测样本为血小板时，出现干扰小脉冲和血小板粒子形成的小脉冲相互混淆的情况，极大的提高了血细胞分析仪的检测精度。
以下通过具体实施例来进一步说明本发明及其优点。
实施例1深度溶血处理检测血小板
按以下配方配制本发明的溶血剂。
Figure PCTCN2019084660-appb-000004
将20微升新鲜血液样本加入到1mL上述配制好的溶液中，在45℃条件下孵育60秒制备得到待测试样，然后用流式细胞仪(迈瑞BriCyte E6)进行测定。收集数据设置增益为500，采集测定角度为90度的侧向散射光获取试样中粒子的侧向散射光强度信息；并收集0度的前向散射光信号。血小板和白细胞的散点分布图如图10所示，从图10可以看出，红细胞碎片、血小板及白细胞的区分度较大，可以非常清晰的对这三团粒子进行区 分，可以同时对血小板和白细胞进行有效的分类统计。通过计算划分白细胞和PLT的比例，结合流式细胞仪的进样量，可以计算出该样本的白细胞和PLT的浓度分别为9.8×10 9/L和166×10 9/L。
用同样的样本，利用贝克曼粒子计数器Z2测得白细胞的浓度为9.6×10 9/L；通过人工镜检计数得出PLT的浓度为171×10 9/L。本发明的方法获得的血小板和白细胞计数与传统方法的测定结果具有较好的一致性。
实施例2对比深度溶血处理和常规溶血处理对血小板的展出情况
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000005
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长488nm，增益为500，采集0°前向散射光强度和90°侧向散射光强度信息，得到二维细胞散点图如图11中A所示。
作为对比，取同一血样的20微升新鲜血液加入到1mL迈瑞血液分析仪BC-6800配套的LD溶血剂(其中含有常规溶血剂)中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长488nm，增益为500，采集应该是0°前向散射光强度和90°侧向散射光强度信息，得到二维细胞散点图如图11中B所示。
对比图11中的A、B两图可明显看出采用本发明方法中对血样进行深度溶血(A)后可明显将血小板(中部的粒子团)和红细胞的碎片区(左下部的粒子团)分开；而用常规溶血剂处理的样本完全无法区分血影与血小板。
实施例3深度溶血并加入核酸染料通过侧向光散射和荧光信号对血小板和白细胞计数
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000006
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长488nm，增益为500，采集90度侧向荧光强度和0度的前向散射光强度信息，得到二维细胞散点图如图12A所示。
从图中可以看出，在溶血状态下，加入核酸染料，能够有效的将血小板和RET在荧光方向有效的区分展出。通过划分PLT散点的比例，以及根据流式细胞仪的进样量，可以计算出该样本的PLT浓度为198×10 9/L。而用人工镜检的方法计算的PLT浓度为202×10 9/L。本实施例的核酸染色方法能有效的使血小板与红细胞碎片区分。
此外，本实施例的方法对白细胞的分类计数没有影响。另用20微升同一血样进行与上述相同的处理，并在同一流式细胞仪中测定，设置激发波长488nm，增益为100000，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到细胞散点分布图，如图12B所示。从图中可以看出，染料在染色PLT的同时，也对白细胞有效染色，并可清晰分群。通过划分WBC散点的比例，结合流式细胞仪的进样量，可以计算出该样本的WBC 浓度为7.86×10 9/L。用传统参考方法结合贝壳曼粒子计数器深度Z2对同一样本测量的WBC浓度为8.01×10 9/L。两种方法间具有较好的一致性。
通过划分图12B中淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、和嗜酸性粒细胞，获得其比例分别为21.2％、3.5％、74.8％和0.5％。在迈瑞6800型血细胞分析上对该样本进行测试后，获得淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、和嗜酸性粒细胞的比例分别为20.8％、3.2％、75.1％和0.9％。从上述结果可以看出，本发明方法对白细胞进行划分与市售血细胞分析仪获得的比例具有较好的相关性。
实施例4深度溶血并加入核酸染料通过光散射和荧光信号对血小板、网织红细胞和白细胞计数
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000007
荧光染料结构式：
Figure PCTCN2019084660-appb-000008
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长633nm，增益为500，采集90度侧向荧光强度信息和0度的前向散射光强度信息，得到二维细胞散点图如图13A所示。
如图所示，RET和PLT分别通过荧光和前向散射光信号可以清晰地区分出来。通过划分PLT及RET散点的比例，结合流式细胞仪的进样量，可以计算出该样本的PLT浓度为165×10 9/L，RET的浓度为18.8×10 9/L。用传统的人工镜检测得PLT的数值为175×10 9/L，RET的浓度为20.1×10 9/L。说明本发明的方法能有效的区分和计数血样中的PLT和RET。
另用20微升同一血样进行与上述相同的处理，并在同一流式细胞仪中测定，设置激发波长633nm，增益为100000，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到细胞散点分布图，如图13B所示。
参见图13B白细胞可以清晰地划分为淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、和嗜酸性粒细胞四类。通过划分WBC散点的比例，结合流式细胞仪的进样量，可以计算出该样本的WBC浓度为8.32×10 9/L。用传统参考方法结合贝壳曼粒子计数器Z2对同一样本测量的WBC浓度为8.45×10 9/L。可见，两种方法间具有较好的一致性。
通过划分淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、嗜酸性粒细胞和幼稚粒细胞，获得其比例分别为15.1％、6.1％、75.5％、2.3％和1.0％。在迈瑞6800型血细胞分析上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、嗜酸性粒细胞和幼稚粒细胞的比例分别为14.8％、5.7％、76.2％、1.9％和1.4％。从上述结果 可以看出，本发明方法对白细胞进行划分与市售血细胞分析仪获得的比例具有较好的相关性。
实施例5深度溶血并加入核酸染料通过光散射和荧光信号检测血小板和白细胞
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000009
荧光染料结构式：
Figure PCTCN2019084660-appb-000010
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长633nm，增益为500，采集90度侧向荧光强度信息和0度的前向散射光强度信息，得到二维细胞散点图如图14A所示。
如图所示，RET和PLT分别通过荧光信号和前向散射光信号可以清晰地区分出来。通过划分PLT及RET散点的比例，结合流式细胞仪的进样量，可以计算出该样本的PLT浓度为189×10 9/L，RET的浓度为101×10 9/L。用传统人工镜检测得PLT的数值为201×10 9/L，RET的浓度为112×10 9/L。说明本发明的方法能有效的区分和计数血样中的PLT和RET。
另用20微升同一血样进行与上述相同的处理，并在同一流式细胞仪中测定，设置激发波长633nm，增益为100000，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到细胞散点分布图，如图14B所示。
白细胞可以清晰划分为淋巴细胞，单核细胞、中性粒细胞及嗜碱性粒细胞、和嗜酸性粒细胞四类。通过划分WBC散点的比例，结合流式细胞仪的进样量，可以计算出该样本的WBC浓度为6.54×10 9/L。用传统参考方法结合贝壳曼粒子计数器Z2对同一样本测量的WBC浓度为6.32×10 9/L。可见，两种方法间具有较好的一致性。
通过划分淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞，和嗜酸性粒细胞的比例分别为19.8％、3.2％、75.8％和1.2％。在迈瑞6800型血细胞分析上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞、和嗜酸性粒细胞的比例分别为20.3％、2.9％、75.1％和1.7％，从从上述结果可以看出，本发明方法对白细胞进行划分与市售血细胞分析仪获得的比例具有较好的相关性。
实施例6深度溶血并加入膜染料通过光散射和荧光信号检测血小板和白细胞
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000011
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长488nm，增益为500，采集90度侧向荧光强度信息和0度的前向散射光强度信息，得到二维细胞散点图如图15所示。
由图15A可见两个深色粒子团(下部及右侧)均为被裂解的红细胞碎片，中间的粒子团为血小板，说明该方法同样能够明显地将血小板和红细胞的碎片区分开。
另用20微升同一血样进行与上述相同的处理，并在同一流式细胞仪中测定，设置激发波长633nm，增益为100000，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到细胞散点分布图。
如图15B所示，通过划分淋巴细胞、单核细胞、中性粒细胞+嗜酸性粒细胞的比例分别为16.4％、6.1％、77.5％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞的比例分别为16％、5.8％、78.2％、1.9％。从上述结果可以看出，本发明方法对白细胞进行划分与市售血细胞分析仪获得的比例具有较好的相关性。
实施例7深度溶血并加入线粒体染料通过光散射和荧光信号检测血小板和白细胞
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000012
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长633nm，增益为500，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到二维细胞散点图如图16所示。
由图16可见两个粒子团，其中右侧为被裂解的红细胞碎片，左侧为血小板，说明该方法同样能够明显地将血小板和红细胞的碎片区分开。
实施例8深度溶血并加入膜染料和核酸染料通过光散射和荧光信号检测血小板和网织红细胞
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000013
将20微升新鲜血液样本加入到1mL按上述配方配制的溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)进行检测。设置激发波长488nm，增益为500，采集90度侧向荧光强度信息和0度的前向散射光强度信息，得到二维细胞散点图如图17所示。
如图17A所示，两个深色粒子团均为被裂解的红细胞碎片，中间的粒子团为血小板，说明该方法能够更加明显地将血小板和红细胞的碎片区分开；最右侧的粒子团识别为网织红细胞，因而该方法能够进一步获得网织红细胞的信息。
另用20微升同一血样进行与上述相同的处理，并在同一流式细胞仪中测定，设置激发波长633nm，增益为100000，采集90度侧向荧光强度信息和90度的侧向散射光强度信息，得到细胞散点分布图。
如图17B所示，通过划分淋巴细胞、单核细胞、中性粒细胞+嗜酸性粒细胞的比例分别为16.8％、6.4％、76.8％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞的比例分别为16％、5.8％、78.2％、1.9％。从上述结果可以看出，本发明方法对白细胞进行划分与市售血细胞分析仪获得的比例具有较好的相关性。
实施例9测试PLT及RET相关性
按以下配方配制本发明的检测试剂。
Figure PCTCN2019084660-appb-000014
荧光染料结构式：
Figure PCTCN2019084660-appb-000015
选取12支新鲜血液样本，每支样本分别取20微升加入到1mL上述溶液中，在45℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6，激发波长633nm，设置增益为500)收集数据。采集90度侧向荧光强度信息和0度的前向散射光强度信息。RET和PLT分别通过荧光信号和前向散射光信号可以区分出来。通过划分PLT及RET散点的比例，以及流式细胞仪的进样量，可以计算出对应样本的PLT浓度和RET的浓度。接着将同样的12支样本放置于迈瑞6800型血细胞分析仪中测试，记录仪器给出的PLT及RET浓度。根据检测数据，分别作出针对RET浓度和PLT浓度用本发明的方法在流式细胞仪上的检测结果与按常规方法在迈瑞6800型血细胞分析仪上的检测结果的相关性图，如图18A和B所示：
从图中可以看出，与迈瑞6800型血细胞分析仪的测试结果相比，采用核酸染料在深度溶血状态下，能够对PLT及RET进行区分染色，并能够对相关粒子进行计数，两种方法的测试结果具有很好的相关性。
实施例10使用染料Alexa Fluor 488分析血样
试剂配制：
第一试剂
Figure PCTCN2019084660-appb-000016
Figure PCTCN2019084660-appb-000017
第二试剂
染料Alexa Fluor 488        30mg
乙二醇                     950ml
甲醇                       50ml
将20微升的新鲜血加入到1mL的上述第一试剂溶液中，并加入20微升的第二试剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长488nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息，细胞散点分布如图19所示。
从图19A中可以看出，在使用膜染料Alexa Fluor 488时，不仅能够将血小板(圈中)与血影有效区分开，同时，该染料对白细胞的染色效果也较好，能够实现对白细胞的分类计数。通过进样量体积以及血小板的测试粒子数，计算出样本的血小板值为216×10 9/L，而通过经典人工镜检的计数方法测量值同样为216×10 9/L。
根据图19B，使用相同的方法对白细胞进行计数，所得结果为8.86×10 9/L，而通过贝克曼粒子计数器Z2测试数值为8.81×10 9/L，两者具有较好的一致性。通过划分淋巴细胞、单核细胞、中性粒细胞和嗜酸性粒细胞的比例分别为17.8％、4.8％、75.8和1.6％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞及嗜碱性粒细胞和嗜酸性粒细胞的比例分别为18.1％、5.1％、74.8％和2.0％，从上述结果可以看出，上述方法对白细胞进行划分与血细胞分析仪中的比例具有较好的相关性。
实施例11使用染料DiD分析血样
试剂配制：
第一试剂
Figure PCTCN2019084660-appb-000018
第二试剂
染料DiD                    30mg
乙二醇                     950ml
甲醇                       50ml
将20微升的新鲜血加入到1mL的上述第一试剂溶液中，并加入20微升第二试剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长633nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息，细胞散点分布如图20所示。
从图20A中可以看出，染料染色后，能够有效地的将血小板和血影区分，通过对其散点数进行划分，进而计算出血小板的浓度为158×10 9/L，而通过经典人工镜检的计数方法测量值为160×10 9/L，因此，染料能够对血小板粒子在溶血状态下进行有效的区分并计数。
根据图20B，使用相同的方法对白细胞进行计数，所得结果为4.35×10 9/L，而通过参考方法结合贝克曼粒子计数器Z2的测量值为4.28×10 9/L，两者具有较好的一致性。通过划分淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞的比例分别为14.8％、6.8％、 74.5％和3.9％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞的比例分别为15.9％、7.2％、72.2％和4.7％，从上述结果可以看出，上述方法对白细胞进行划分与血细胞分析仪中的比例具有较好的相关性。
实施例12使用染料罗丹明123分析血样
试剂配制：
第一试剂
Figure PCTCN2019084660-appb-000019
第二试剂
染料罗丹明123               30mg
乙二醇                      950ml
甲醇                        50ml
将20微升的新鲜血加入到1mL的上述第一试剂溶液中，并加入20微升第二试剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长488nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息，细胞散点分布如图21A所示。
从图21A中可以看出，染料罗丹明123染色后，能够有效地的将血小板和血影区分，通过对其散点数进行划分，进而计算出血小板的浓度为208×10 9/L，而通过经典人工镜检的计数方法测量值为201×10 9/L，因此，染料罗丹明123能够对血小板粒子在溶血状态下进行有效的区分并计数。
根据图21B，使用相同的方法对白细胞进行计数，所得结果为4.02×10 9/L，而通过参考方法结合贝克曼粒子计数器Z2的测量值为3.98×10 9/L，两者具有较好的一致性。通过划分淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞的比例分别为15.9％、7.1％、71.5％和5.5％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞的比例分别为15.9％、6.8％、72.2％和5.1％，从上述结果可以看出，上述方法对白细胞进行划分与血细胞分析仪中的比例具有较好的相关性。
实施例13使用染料Mitotracker Deep Red分析血样
试剂配制：
第一试剂
Figure PCTCN2019084660-appb-000020
第二试剂
染料Mitotracker Deep Red    30mg
乙二醇                      950ml
甲醇                        50ml
将20微升的新鲜血加入到1mL的上述第一试剂溶液中，并加入20微升的第二试 剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长633nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息，细胞散点分布如图22所示。
从图22A中可以看出，在使用Mitotracker Deep Red时，能够将血小板(图中虚线范围)与血影区分开，其中，浅色散点表示为血小板，通过对其散点数进行划分，进而计算出PLT的浓度为165×10 9/L，而通过经典人工镜检计数的方法对其进行计数，其浓度为201×10 9/L，可以看出染料Mitotracker Deep Red也能将血小板区分出来。
根据图22B，使用相同的方法对白细胞进行计数，所得结果为5.12×10 9/L，而通过参考方法结合贝克曼粒子计数器Z2的测量值为5.08×10 9/L，两者具有较好的一致性。通过划分淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞的比例分别为14.8％、7.8％、74.5％和2.9％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞的比例分别为15.9％、7.2％、73.2％和3.7％，从上述结果可以看出，上述方法对白细胞进行划分与血细胞分析仪中的比例具有较好的相关性。
实施例14使用染料Mitotracker Red分析血样
试剂配制：
第一试剂
Figure PCTCN2019084660-appb-000021
第二试剂
染料Mitotracker Red        30mg
乙二醇                     950ml
甲醇                       50ml
将20微升的新鲜血加入到1mL的上述第一试剂溶液中，并加入20微升的第二试剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长488nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息，细胞散点分布如图23所示。
从图23A可以看出，在使用染料Mitotracker Red时，不仅能够较为有效的将血小板(图中虚线范围)和血影区分，同时从图23B可以看出在该染料对白细胞的染色性能上，也能够有效的对白细胞进行分类，从而实现了在检测白细胞的同时，对血小板进行了有效的分类计数。通过进样量体积以及血小板的测试粒子数，计算出样本的血小板值为176×10 9/L，而通过经典人工镜检的计数方法测量值为172×10 9/L，两者具有较好的一致性。
根据图23B，使用相同的方法对白细胞进行计数，所得结果为7.65×10 9/L，而通过参考方法结合贝克曼粒子计数器Z2的测量值为7.73×10 9/L，两者具有较好的一致性。通过划分淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞和幼稚粒细胞的比例分别为13.7％、6.1％、77.5％、0.7％和2.0％，而在迈瑞6800血细胞分析仪上对该样本进行测试后，淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞和幼稚粒细胞的比例分别为14.1％、5.8％、76.2％、1.9％和2.0％，从上述结果可以看出，上述方法对白细胞进行划分与血细胞分析仪中的比例具有较好的相关性。
实施例15以实施例10为例验证血小板测试的相关性
试剂配制：同实施例10。
取将20支新鲜血样本，每支血样的测试方法如下：取20微升血样加入到上述配置第一试剂溶液1mL中，并加入20微升第二试剂，在35℃条件下孵育60秒后用流式细胞仪(迈瑞BriCyte E6)收集数据(激发波长488nm)设置增益为500，采集90度侧向荧光信号收集荧光染色信息，并采用测定角度为0度的前向散射光强度信息。通过划分出血小板的散点，结合流式细胞仪中的进样量，计算出血样的血小板浓度，测试完毕后，将血样在迈瑞6800仪器上进行测试，记下其血小板的测试值，用两种测试方法所得的结果作出相关性曲线：
从图24中可以看出，使用膜染料Alexa Fluor 488在测试血小板时，其测试数值与迈瑞6800仪器测试值具有较好的一致性，在100×10 9/L～250×10 9/L的数值范围内，相关系数为0.9817。
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 (44)

  1. 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其光散射特性显著不同于血小板的碎片；
    使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信息；和
    根据所述光学信息中的至少两种获得血小板的光学信息。
  2. 根据权利要求1所述血液检测方法，其中所述至少两种光学信息为前向散射光强度和侧向散射光强度以将血小板与裂解的红细胞碎片区分开。
  3. 根据权利要求2所述血液检测方法，所述方法进一步包括根据前向散射光强度和侧向散射光强度的光学信息获得白细胞的光学信息，优选地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。
  4. 根据权利要求1所述血液检测方法，其中所述溶血剂为选自烷基糖苷、三萜皂苷、甾族皂苷中的至少一种。
  5. 根据权利要求4所述血液检测方法，其中所述烷基糖苷选自具有通式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。
  6. 根据权利要求1所述的血液检测方法，其中所述第一试剂进一步包括：
    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 PCTCN2019084660-appb-100001
    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。
  7. 根据权利要求1所述的血液检测方法，所述方法进一步包括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。
  8. 根据权利要求7所述的血液检测方法，其中使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信息进一步包括荧光信息，并且根据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。
  9. 根据权利要求7或8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第一荧光染料。
  10. 根据权利要求9所述的血液检测方法，其中所述膜特异性染料选自DiA、DiD、DiI、DiO、DiR、DiS、FDA、Alexa Fluor 488、Super Fluor 488及以它们为母体的变形结构所组成的组，以及所述线粒体特异性染料选自Janus Green B、MitoLite Red、罗丹明123和Mitotracker系列及以它们的母体所组成的组。
  11. 根据权利要求9或10所述的血液检测方法，其中根据荧光强度和前向散射光强度信息识别血小板。
  12. 根据权利要求9或10所述的血液检测方法，其中所述方法进一步包括：当根据前向散射光强度和荧光强度构成的散点图的预设区域中的粒子数超过预定阈值时报警网织红细胞。
  13. 根据权利要求7或8所述的血液检测方法，其中所述荧光染料包括选自核酸特 异性染料中的一种的第二荧光染料，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。
  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：根据荧光强度和散射光强度信息识别网织红细胞，优选的，根据荧光强度和前向散射光强度信息区分血小板和网织红细胞。
  15. 根据权利要求14所述的血液检测方法，其中所述方法进一步包括：根据荧光强度和前向散射光强度信息对网织红细胞进行计数。
  16. 根据权利要求7或8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的第一荧光染料和选自核酸特异性染料中的一种的第二荧光染料。
  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：根据荧光强度和侧向散射光强度信息区分血小板和网织红细胞。
  18. 根据权利要求1～17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。
  19. 根据权利要求1～18中任一项所述的血液检测方法，其中所述光学检测系统，包括：光学子系统、流动室、第一检测器；
    所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，
    所述激光器，配置为发射激光光束；
    所述前光组件，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一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
    所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
    所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光为所述激光光束经所述流动室所产生。
  20. 根据权利要求19所述的血液检测方法，其中所述光隔离器由采用粘合方式相互连接的分光棱镜及偏振态转换组件构成；
    所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
    所述偏振态转换组件，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射。
  21. 如权利要求19所述的血液检测方法，其中所述光隔离器由采用粘合方式相互连接的检偏器及偏振态转换组件组成；
    所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
    所述偏振态转换组件，配置为改变经所述检偏器的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光的反射光偏振态为S偏振光而被所述检偏器隔离。
  22. 如权利要求19所述的血液检测方法，所述光隔离器由采用粘合方式相互连接的带通滤光片及倍频晶体组成；
    所述带通滤光片，配置为使波长为λ的所述激光光束通过；
    所述倍频晶体，配置为对经所述带通滤光片的激光光束进行倍频，并对所述倍频后的激光光束的反射光再次进行倍频，而被所述带通滤光片滤除。
  23. 如权利要求19所述的血液检测方法，其中所述前光组件还包括准直透镜；
    所述准直透镜，沿所述激光光束的传播方向设置于所述激光器与所述光隔离器之间，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。
  24. 如权利要求19所述的血液检测方法，其中所述后光组件还包括挡直光阑；
    所述前光组件，还配置为对所述激光光束进行前光处理，使得经所述前光处理的激光光束在第二方向上汇聚于所述挡直光阑处。
  25. 如权利要求19所述的血液检测方法，其中所述前光组件还包括第一光汇聚组件及第二光汇聚组件；
    所述第一光汇聚组件，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所述流动室的血细胞被测样本处并产生散射光；
    所述第二光汇聚组件，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所述后光组件包括的挡直光阑处。
  26. 如权利要求19所述的血液检测方法，其中所述后光组件还包括第三汇聚组件及小孔光阑；
    所述第三汇聚组件，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，并经所述小孔光阑的小孔进入所述第一检测器。
  27. 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，所述溶血剂裂解所述血液样本中的红细胞；
    使待测试样中的粒子逐个通过光学检测系统的检测区，获取所述待测试样的光学信息；和
    根据所述光学信息中的至少两种光学信息获得血小板的光学信息。
  28. 根据权利要求27所述的血液检测方法，其中所述溶血剂将所述血液样本中的红细胞裂解为其光散射特性显著不同于血小板的碎片。
  29. 根据权利要求28所述的血液检测方法，其中所述方法根据前向散射光强度和侧向散射光强度对血小板计数。
  30. 根据权利要求28或29所述的血液检测方法，其中所述方法进一步根据前向散射光强度和侧向散射光强度将白细胞区分为至少包括单核细胞、淋巴细胞和中性粒细胞的亚群。
  31. 根据权利要求27所述的血液检测方法，所述方法进一步包括：用第二试剂对血液样本进行处理其中所述第二试剂包含选自膜特异性染料和线粒体特异性染料中的一种荧光染料，或者包含选自核酸特异性染料中的一种荧光染料，以进一步获得荧光信号，根据散射光强度和荧光强度信息区分血小板和网织红细胞。
  32. 根据权利要求31所述的血液检测方法，其中所述方法进一步包括：根据荧光强度和侧向散射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。
  33. 根据权利要求31所述的血液检测方法，其中根据荧光强度和前向散射光强度信息全区分血小板。
  34. 根据权利要求31所述的血液检测方法，其中所述方法进一步包括：当根据前向散射光强度和荧光强度构成的散点图的预设区域中的粒子数超过预定阈值时报警网织红细胞。
  35. 根据权利要求27所述的血液检测方法，其中，所述方法进一步包括：用第二试剂对血液样本进行处理，其中所述第二试剂包含选自膜特异性染料和线粒体特异性染料中的一种荧光染料，和选自核酸特异性染料中的一种荧光染料，获得荧光信号，根据散射光强度和荧光强度信息区分血小板和网织红细胞。
  36. 根据权利要求35所述的血液检测方法，其中所述方法进一步包括：根据荧光 强度和前向散射光强度信息区分血小板和网织红细胞，优选地进一步对网织红细胞计数。
  37. 根据权利要求27中任一项所述的血液检测方法，其中所述光学检测系统，包括：光学子系统、流动室、第一检测器；
    所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，
    所述激光器，配置为发射激光光束；
    所述前光组件，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一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
    所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
    所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光为所述激光光束经所述流动室所产生。
  38. 根据权利要求37所述的血液检测方法，其中所述光隔离器由采用粘合方式相互连接的分光棱镜及偏振态转换组件构成；
    所述分光棱镜，配置为反射入射的所述激光光束的S偏振分量，透射入射的所述激光光束的P偏振分量；
    所述偏振态转换组件，配置为改变经所述分光棱镜透射的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光反射后的偏振态为S偏振光而被所述分光棱镜反射；或者
    其中所述光隔离器由采用粘合方式相互连接的检偏器及偏振态转换组件组成；
    所述检偏器，配置为仅允许所述激光光束的P偏振分量通过；
    所述偏振态转换组件，配置为改变经所述检偏器的所述P偏振分量的偏振态，使所述P偏振分量从线偏振光变成圆偏振光，并改变所述圆偏振光的反射光偏振态为S偏振光而被所述检偏器隔离。
  39. 如权利要求27所述的血液检测方法，所述两种光学信息为前向散射光强度信息和侧向散射光强度信息，根据所述前向散射光强度信息和侧向散射光强度信息对血小板进行计数和/或将白细胞至少区分为单核细胞、淋巴细胞和中性粒细胞亚群。
  40. 一种血液分析系统，包括：
    采样部，用于获取血液样本，并将所述血液样本输送到所述反应部；
    试剂供应部，用于贮存至少第一试剂并根据需要供应到所述反应部；
    反应部，包括混合室，用于将所述血液样本与第一试剂混合以形成待测试样，其中所述第一试剂包括溶血剂，所述溶血剂裂解所述血液样本中的红细胞；
    光学检测系统，包括流动室和至少第一检测器，用于当所述待测试样由所述混合室被输送到所述光学系统并使所述待测试样中的粒子逐个通过流动室到达检测区时，所述第一检测器对待测试样中的粒子进行检测以获得所述待测试样中的光学信息；和
    数据处理模块，其与所述光学系统可操作地连接，并包括处理器和存储有计算机程序的非暂时性计算机可读存储介质，其中当所述计算机程序被所述处理器执行时，执行以下步骤：根据所述光学信息中的至少两种光学信息获得血小板的光学信息。
  41. 根据权利要求40所述的血液分析系统，其中所述溶血剂将所述血液样本中的红细胞裂解为其光散射特性显著不同于血小板的碎片，且其中所述处理模块中，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：
    根据所获得的光学信息中的前向散射光强度和侧向散射光强度对血小板计数；和/或
    根据前向散射光强度和侧向散射光强度将白细胞至少区分为单核细胞、淋巴细胞和 中性粒细胞亚群。
  42. 根据权利要求40所述的血液分析系统，其中，
    所述混合室用于所述血液样本与第二试剂混合以形成待测试样，其中所述第二试剂包含选自膜特异性染料和线粒体特异性染料中的一种荧光染料；
    所述光学检测系统包括第二检测器，所述第二检测器为荧光检测器，以便当所述待测试样中的粒子逐个通过检测区时进一步获得荧光信号；和
    所述数据处理模块中，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：
    根据荧光强度和侧向散射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；
    根据荧光强度和前向散射光强度信息全区分血小板；和/或
    当根据前向散射光强度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报警网织红细胞。
  43. 根据权利要求42所述的血液分析系统，其中所述第二试剂进一步包含选自核酸特异性染料中的一种荧光染料，和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：根据荧光强度和散射光强度信息区分血小板和网织红细胞，优选地进一步对网织红细胞计数。
  44. 根据权利要求40所述的血液分析系统，其中所述光学检测系统，进一步包括：光学子系统，
    所述光学子系统包括：激光器、前光组件及后光组件，所述前光组件包括光隔离器；其中，
    所述激光器，配置为发射激光光束；
    所述前光组件，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一方向上汇聚于所述流动室的血细胞被测样本处并产生散射光；
    所述后光组件，沿所述激光光束的传播方向设置于所述流动室之后，配置为对所述散射光进行后光处理，使得经所述后光处理的所述散射光进入所述第一检测器进行光强检测；
    所述光隔离器，配置为将反射光与所述激光器隔离；所述反射光为所述激光光束经所述流动室所产生。
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