WO2021077327A1 - 分析血液样本中红细胞方法及血液分析系统 - Google Patents

分析血液样本中红细胞方法及血液分析系统 Download PDF

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Publication number
WO2021077327A1
WO2021077327A1 PCT/CN2019/112766 CN2019112766W WO2021077327A1 WO 2021077327 A1 WO2021077327 A1 WO 2021077327A1 CN 2019112766 W CN2019112766 W CN 2019112766W WO 2021077327 A1 WO2021077327 A1 WO 2021077327A1
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suspension
sample
volume distribution
distribution information
red blood
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PCT/CN2019/112766
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English (en)
French (fr)
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叶波
祁欢
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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 PCT/CN2019/112766 priority Critical patent/WO2021077327A1/zh
Priority to CN201980100664.6A priority patent/CN114450589A/zh
Publication of WO2021077327A1 publication Critical patent/WO2021077327A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the invention relates to the field of blood cell detection, in particular to a red blood cell analysis method and an analysis system thereof.
  • the blood cell analyzer is an instrument that can detect cell-related information in the blood. It can count and classify white blood cells (WBC), red blood cells (RBC), platelets (PLT) and other cells.
  • WBC white blood cells
  • RBC red blood cells
  • PHT platelets
  • the most commonly used method is the small hole electrical impedance method.
  • this method when the cell particles in the blood pass through the small hole, it causes an instantaneous voltage change to form a voltage pulse.
  • the intensity of the voltage pulse reflects the volume of the cells, and the number reflects the number of cells passing through the micropores. From this, a histogram of the volume distribution of the measured blood cells is obtained. Through the histogram, platelets and red blood cells can be distinguished.
  • the blood cell analyzer using the small hole impedance method can also obtain the commonly used red blood cell parameters through the histogram distribution parameters of the red blood cells, such as the mean corpuscular volume (MCV, Mean Corpuscular Volume), and the average corpuscular hemoglobin content (MCH, Mean Corpuscular Hemoglobin), Mean Corpuscular Hemoglobin Concentration (MCHC, Mean Corpuscular Hemoglobin Concentration), Red Blood Cell Distribution Width Coefficient of Variation (RDW-CV, Red Blood Cell Distribution Width Coefficient of Variation), Standard Deviation of Red Blood Cell Distribution Width (RDW-SD) , Red Blood Cell Distribution Width Standard Deviation), Hematocrit (HCT, Hematocrit), etc.
  • MCV Mean Corpuscular Volume
  • MHC Mean Corpuscular Hemoglobin Concentration
  • RDW-CV Red Blood Cell Distribution Width Coefficient of Variation
  • Standard Deviation of Red Blood Cell Distribution Width RDW-SD
  • Red Blood Cell Distribution Width Standard Deviation
  • red blood cells there may be a larger volume of red blood cells in the red blood cells, and some white blood cells such as small lymphocytes may also have a small volume, which makes the two types indistinguishable on the histogram.
  • the number of white blood cells in the blood is much lower than that of red blood cells, and the difference between the numbers of the two is about 1000 times. Therefore, the influence of a small amount of white blood cells on the red blood cell count, hemoglobin content HGB, MCV and other parameters can be almost ignored.
  • the white blood cells exceed 40.0 ⁇ 10 ⁇ 9/L, the white blood cells will affect the analysis of hemoglobin content, and will also falsely increase the red blood cell count, MCV and other morphological parameters. Eventually, the red blood cell count and the calculation results of related parameters are inaccurate.
  • the whole blood sample can be centrifuged in a packed tube at low speed to remove the white blood cell layer and plasma layer, and then add an equal amount of diluent to mix and then use the analyzer for detection. This obviously increases the complexity of detection and cannot meet the requirements for rapid analysis of blood.
  • Zeng Sugen et al. (Laboratory Medicine, 2010, 25(3): 244-246) also reported that certain diseases (such as leukemia) caused abnormally high levels of leukemia that would interfere with the red blood cell parameters of all models of blood analyzers, and gave a A simple correction method.
  • This method is to directly subtract the white blood cell count from the original red blood cell count to obtain the corrected red blood cell count.
  • this method can only obtain the count of red blood cells, but cannot obtain all the morphological parameters of red blood cells, and the actual application range is too narrow.
  • a method for analyzing red blood cells in a sample includes the following steps:
  • a blood analysis system includes: a sampling part, a reagent supply part, a reaction part, a detection part, a controller and a processor, wherein:
  • the sampling part is configured to obtain at least two samples from a blood sample, and to transport the samples to the reaction part respectively;
  • the reagent supply part is configured to supply required reaction reagents to the reaction part
  • the reaction part includes a first reaction chamber for mixing a first sample with a first reaction reagent to prepare a first suspension; and a second reaction chamber for mixing a second sample with a second reaction reagent Preparing a second suspension, and the reaction part is configured to supply the first suspension and the second suspension to the detection part respectively;
  • the detection part includes an electrical detection part and an optical detection part, wherein the electrical detection part is configured to make the particles in the first suspension pass through the resistance detector one by one to obtain the first electrical signal of the particles in the first suspension , And the optical detection part is configured to make the particles in the second suspension pass through the optical detector one by one to obtain the optical information of the particles in the second suspension;
  • the controller is coupled to the sampling part, the reagent supply part, the reaction part, and the detection part, and controls the actions of the sampling part, the reagent supply part, the reaction part, and the detection part;
  • the processor, the processor and the detection unit are coupled, wherein
  • the processor obtains the electrical signal and the optical information from the detection unit, obtains first volume distribution information according to the electrical signal, obtains white blood cell distribution information according to the optical information, and obtains the information by using forward scattered light intensity Second volume distribution information, and using the second volume distribution information to correct the first volume distribution information to obtain corrected volume distribution information of red blood cells in the blood sample.
  • the blood analysis system includes:
  • Sampling part, reagent supply part, reaction part, detection part and controller including:
  • a sampling part configured to obtain at least two samples from a blood sample, and to transport the samples to the reaction part respectively;
  • a reagent supply part configured to supply required reaction reagents to the reaction part
  • the reaction part includes a first reaction chamber for mixing a first sample with a first reaction reagent to prepare a first suspension, and a second reaction chamber for mixing a second sample with a second reaction reagent to prepare a first suspension Two suspensions, and the reaction part is configured to supply the first suspension and the second suspension to the detection part respectively;
  • the detection part includes an electrical detection part, wherein the electrical detection part is configured to make the particles in the suspension pass through the resistance detector one by one to obtain the first electrical signal of the first suspension and the second suspension. Second electrical signal
  • a controller which is coupled to the sampling part, the reagent supply part, the reaction part, and the detection part, and controls the actions of the sampling part, the reagent supply part, the reaction part, and the detection part;
  • a processor, the processor and the detection unit are coupled, wherein
  • the processor obtains the first and second electrical signals from the detection unit, obtains first volume distribution information based on the first electrical signal, obtains third volume distribution information based on the second electrical signal, and uses The third volume distribution information corrects the first volume distribution information to obtain corrected volume distribution information of red blood cells of the blood sample.
  • a computer-readable storage medium with a computer program stored on the computer-readable storage medium, characterized in that the computer program implements the following steps when executed by a processor:
  • the first electrical signal obtained by detecting the first suspension and obtain the first volume distribution information according to the first electrical signal, wherein the first suspension is composed of a first sample obtained from a blood sample and a second A reaction reagent is mixed, and the electrical signal is obtained by passing the first suspension through the detection of the electrical detection part;
  • the optical information obtained by detecting the second suspension obtain the white blood cell distribution information from the optical information, and obtain the second volume distribution information by using the forward scattered light intensity, wherein the second suspension is obtained from the A second sample obtained from a blood sample is mixed with a second reaction reagent containing at least a hemolytic agent, and the optical information is obtained by passing the second suspension through an optical detection unit for detection,
  • the blood cell analysis method of the present invention utilizes the existing white blood cell (hemolysis) optical detection channel or only the white blood cell (hemolysis) electrical impedance detection channel, which can effectively exclude the sample, especially the sample with high white blood cell content, the white blood cell to the red blood cell analysis In order to obtain accurate red blood cell count and related parameters, so as to provide accurate test information for clinical diagnosis and treatment.
  • WBC/BASO channels, DIFF channels, NRBC channels, and electrical impedance detection channels can usually be used to detect white blood cells and obtain count values.
  • the following detailed example demonstrates that no matter which channel the white blood cell volume distribution information is used to correct the white blood cell and red blood cell volume distribution information obtained by the electrical impedance method, accurate red blood cell volume distribution information can be obtained.
  • white blood cell (hemolytic) electrical impedance detection channel by detecting the hemolyzed sample, white blood cell volume distribution information can also be obtained, which is used to correct the volume distribution information including white blood cells and red blood cells, and obtain accurate red blood cell volume distribution information.
  • the method and analysis system of the present invention can obtain accurate red blood cell count and related morphological parameters in a simple method.
  • Fig. 1A is a schematic flow chart of the method steps for analyzing red blood cells in a blood sample according to the first embodiment of the present invention
  • Fig. 1B is a schematic flow chart of the method steps for analyzing red blood cells in a blood sample according to the second embodiment of the present invention
  • FIG. 2A is a schematic block diagram of the structure of the blood analysis system according to the first embodiment of the present invention.
  • FIG. 2B is a schematic block diagram of the structure of a blood analysis system according to a second embodiment of the present invention.
  • Figure 3 is a first histogram of the first test solution according to Example 1;
  • FIG. 5 is an overlapping view of the first histogram and the second histogram according to Embodiment 1, where A is the full image, and B is an enlarged view of the 100-300fL area;
  • Fig. 6 is a corrected red blood cell histogram according to Example 1.
  • Figure 7 is a first histogram of the first test solution according to Example 2.
  • FIG. 9 is an overlapping view of the first histogram and the second histogram according to Embodiment 2, where A is the full image, and B is an enlarged view of the 100-300fL area;
  • Example 10 is a corrected red blood cell histogram according to Example 2.
  • Figure 11 is a first histogram of the first test solution according to Example 3.
  • FIG. 13 is an overlapping view of the first histogram and the second histogram according to Embodiment 3, where A is the full image, and B is an enlarged view of the 100-300fL area;
  • Example 15 is a first histogram of the first test solution according to Example 4.
  • 16 is a scatter diagram of the white blood cell particle group of the second test solution according to Example 4.
  • FIG. 17 is an overlapping view of the first histogram and the second histogram according to Embodiment 4, where A is the full image, and B is an enlarged view of the 150-300fL area;
  • Figure 19 is a first histogram of the first test solution according to Example 5.
  • 21 is an overlapping view of the first histogram and the second histogram according to Embodiment 5, where A is the full image and B is an enlarged view of the 100-200fL area;
  • Figure 22 is a corrected red blood cell histogram according to Example 5.
  • FIG. 23 is a histogram of red blood cells obtained by direct testing according to Comparative Example 1.
  • FIG. 23 is a histogram of red blood cells obtained by direct testing according to Comparative Example 1.
  • the term "histogram” mentioned in this context is a graphical form of cell volume distribution and a common form of continuous variable probability distribution.
  • the cell volume distribution can also be presented in digital form in a table or list with the same or similar resolution as the volume histogram, or presented in any other suitable manner known in the art.
  • the present invention provides a simple red blood cell analysis method that removes white blood cell interference.
  • the content of white blood cells is only one-thousandth of that of red blood cells, and the volume of white blood cells is usually larger than that of red blood cells.
  • the influence of white blood cells is negligible.
  • the histogram obtained by the conventional impedance method is mixed with a considerable number of white blood cell particles within the volume of red blood cells, which affects the red blood cell count and other morphological analysis results, and further Adversely affect the correct diagnosis.
  • the present invention utilizes the existing channels and detection equipment in the blood analyzer to analyze and obtain the histogram of white blood cells, and correct the histogram of red blood cells detected according to the conventional impedance method according to the histogram of white blood cells, thereby obtaining the Corrected histograms with similar real red blood cell information, and further obtain correct red blood cell counts and other morphological analysis results.
  • a first sample is obtained from the blood sample, the first sample is mixed with a first reaction reagent to obtain a first suspension, and a second sample is obtained from the blood sample so that the second sample Mixing with a second reaction reagent containing a hemolytic agent to obtain a second suspension, wherein in the first suspension, the red blood cells basically maintain their original form, and in the second suspension, the red blood cells are lysed;
  • the second volume distribution information is used to correct the first volume distribution information to obtain the corrected volume distribution information of the red blood cells in the blood sample.
  • a blood sample S11 is provided.
  • the blood sample of the present invention is usually a whole blood sample. It can be a whole blood sample from a mammal, especially a primate, and more specifically a human.
  • a first sample is obtained from the blood sample and mixed with a first reaction reagent S22 to obtain a first suspension.
  • the first reaction reagent is usually a diluent.
  • the present invention has no particular limitation on the diluent, and any diluent suitable for electrical impedance detection can be used in the present invention.
  • the diluent usually includes one or more salts, such as alkali metal salts, and is adjusted to be isotonic to maintain red blood cell volume.
  • the present invention has no particular limitation on the diluent, and any diluent suitable for electrical impedance detection can be used in the present invention.
  • any suitable commercial blood diluent can be used, such as M-68DS diluent and M-53D diluent of Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Shenzhen, China).
  • the first suspension obtained by dilution that is, the first test solution
  • the electrical impedance method may be, for example, the small hole impedance method, the sheath current impedance method, and the like.
  • red blood cells and platelets can be distinguished, and the red blood cell histogram is used as the first histogram. Simultaneously, or sequentially, obtaining a second sample from the blood sample and mixing with a second reaction reagent S32 to obtain a second suspension.
  • the first sample may be subjected to electrical impedance detection and optical detection sequentially.
  • the second sample is obtained by further mixing the first sample after the electrical impedance test with the second test reagent. That is, the first blood sample is mixed with the first detection reagent to obtain the first sample, and the first sample is further mixed with the second detection reagent after the electrical impedance detection to obtain the second sample.
  • the second reaction reagent of the present invention contains a hemolytic agent to dissolve red blood cells in the second sample.
  • the hemolytic agent can be any one or a combination of cationic, nonionic, anionic, and amphiphilic surfactants.
  • the hemolytic agent used to dissolve red blood cells in the second sample is not particularly limited. Therefore, any lysis reagent suitable for the classification of leukocytes in a blood analyzer can be used.
  • the lysis reagent used for the classification of leukocytes in the blood analyzer is usually an aqueous solution containing one or more of the above-mentioned hemolytic agents.
  • the second reaction reagent for processing the second sample further contains a fluorescent dye for staining nucleated blood cells, so that the intensity of scattered light and fluorescence can be further checked.
  • White blood cells are separated and/or nucleated blood cells are distinguished.
  • the dissolving reagent formulation described in U.S. Patent No. 8,367,358 can be used, the entire disclosure of which is incorporated herein by reference.
  • the dissolving reagent disclosed in US Pat. No. 8,367,358 includes a cationic cyanine compound (a fluorescent dye), a cationic surfactant, a nonionic surfactant, and an anionic compound.
  • the dissolving reagent can be used To lyse red blood cells and use fluorescence and light scattering measurements to classify white blood cells into their subgroups.
  • Other suitable fluorescent dyes can also be used in the dissolving reagent.
  • the fluorescent dye may be contained in a separate dyeing solution, and used as a third reaction reagent together with a second reaction reagent that does not contain a fluorescent dye as a dissolving reagent.
  • the staining solution may be added to the blood sample before, after, or at the same time as the dissolving reagent to stain the nucleated blood cells.
  • step S33 the second suspension is further subjected to optical detection, so as to obtain optical information of blood cells in the second suspension.
  • step S34 the optical information of the cells in the second suspension can be obtained from the optical information; then the white blood cell particle group is distinguished according to the optical characteristics of the particles, and further according to the forward scattered light intensity of the white blood cells (which can reflect the cell volume information) The histogram of the white blood cell volume is obtained as the second histogram.
  • Any conventional method can be used to obtain the scatter diagram from the optical information in order to identify the white blood cell particle group.
  • Two-dimensional scattergrams are usually obtained by two kinds of optical information, such as the light intensity of forward and side scattered light, the light intensity of forward scattered light and fluorescent light, the light intensity of side scattered light and fluorescent light, and so on.
  • three-dimensional scatter plots can also be obtained through three kinds of information.
  • the present invention is not particularly limited to this.
  • the volume distribution information of the white blood cells can be obtained through analysis according to the forward scattered light intensity of the white blood cells, and expressed in the form of, for example, a histogram.
  • the method for converting the optical information into a histogram is not particularly limited, and may be any existing suitable method.
  • the forward light scattering signal is used to calculate the one-dimensional volume distribution of particles, that is, the histogram.
  • histogram of the scatter graph in the present invention can also adopt any of the following methods.
  • One method is to use the forward light scattering information of the particles to calculate the derived volume Vol f1 of each particle according to the following formula (1).
  • FSC is the forward scattered light intensity of an individual event in the particle group to be converted (specifically a single white blood cell in the present invention) in the two-dimensional scatter diagram
  • ⁇ is a constant, and its value is related to the detection device
  • Different detection equipment can have different ⁇ values.
  • Another method is to use the forward light scattering information of the particles to calculate the derived volume Vol f2 of each particle according to formula (2).
  • the FSC is also the forward scattered light intensity of an individual event in the particle group to be converted (specifically a single white blood cell in the present invention) in the two-dimensional scatter diagram, and ⁇ and ⁇ are constants. Similarly, its value is related to the detection equipment, and the ⁇ and ⁇ values of different detection equipment can be different.
  • a volume distribution histogram related to the white blood cell volume in the histogram obtained by the DC impedance method can be obtained.
  • the present invention can use the white blood cell information obtained by optical detection to correct the mixed information of red blood cells and white blood cells detected by the electrical impedance method, and thereby obtain accurate red blood cell information.
  • the white blood cell optical information obtained can be used to correct the electrical impedance method after being converted into a histogram.
  • the obtained histogram also verify that the obtained corrected red blood cell histogram is consistent with the histogram result obtained by the electrical impedance method after the blood sample is processed in advance to remove the white blood cells.
  • the method of the present invention can obtain information on the volume distribution of red blood cells, thereby further obtaining relevant red blood cell morphological distribution data, and is not limited to counting red blood cells. Correction.
  • the histogram obtained after correcting the first histogram with the second histogram can obtain the red blood cell count and other morphological parameters (step S16).
  • the other morphological parameters may be selected from at least one parameter in the following group: average red blood cell volume (MCV), average red blood cell hemoglobin content (MCH), average red blood cell hemoglobin concentration (MCHC), coefficient of variation of red blood cell distribution width (RDW-CV), Standard deviation of red blood cell distribution width (RDW-SD), hematocrit (HCT).
  • the method of the present invention does not change the principle of signal detection, and can be applied to a variety of detection occasions.
  • the method of the present invention can utilize various hemolysis samples in a blood analyzer to detect samples. Therefore, when the blood cell analyzer is used for blood sample detection, while obtaining the correct red blood cell related parameters, any required other parameters of the blood sample that can be obtained by conventional electrical impedance method and/or optical detection can be obtained. For example: According to the light intensity of lateral and forward light scattering, basophils and other white blood cell particles can be distinguished; according to the light intensity and fluorescence intensity of forward light scattering, basophils and other white blood cell particles can also be distinguished.
  • nucleic acid substances such as nucleated red blood cells
  • white blood cells classify white blood cells into four categories based on the light intensity and fluorescence intensity of side light scattering, distinguishing eosinophils, neutrophils, monocytes and lymphocytes, but Not limited to this.
  • the method of the present invention further includes the step of outputting the required information and/or parameters.
  • the required information and/or parameters for labeling.
  • the white blood cell count is abnormal (for example, exceeding the first threshold), or according to whether the difference of the red blood cell count before and after the correction exceeds the normal range (for example, exceeding the second threshold), the output information obtained after correction (for example, Histogram) and/or parameters (such as red blood cell count and other morphological parameters) for labeling.
  • the method of the present invention can not only output the corrected histogram and related parameters, but also output the first histogram before correction, and even superimpose the first histogram of the second or third histogram used for correction.
  • the normal range of each item can be further output as a reference.
  • FIG. 2A a schematic diagram of the structure of the blood analyzer 100 that can be used in the above method is shown.
  • the blood analyzer 100 conventionally includes a sampling part 110, a reagent supply part 120, a reaction part 130, a detection part 140, a controller 150, and a processor 160. Of course, it can also include other necessary devices, such as a display unit, an output and an input unit, a housing, and necessary pumps and a hydraulic system, etc. These devices are not described here.
  • the sampling part 110 may include a liquid suction device, a sample divider, etc., to suck the first and second samples for detection from the blood sample, and supply the first sample to the first reaction chamber 131 of the reaction part 130, And the second sample is supplied to the second reaction chamber 132.
  • the reagent supply part 120 is used to supply required reaction reagents to the reaction part.
  • FIG. 2A shows a reagent supply part 120 having two liquid storage tanks according to a specific embodiment: a liquid storage tank A121 and a liquid storage tank B122, which are respectively used to store the first reaction reagent and the second reaction as described above.
  • the reagents are supplied to the first reaction chamber 131 of the reaction part 130, and the second sample is supplied to the second reaction chamber 132 to be mixed with the first and second samples respectively to obtain the first suspension Liquid and a second suspension.
  • the reagent supply part 120 may also have more liquid storage tanks.
  • the reagent storage portion 120 may also have a third reservoir to separately store the fluorescent staining solution.
  • the reagent supply part may not have a reservoir.
  • the reagent supply part sucks and supplies the required reaction reagents from the reagent barrel outside the machine to the reaction part.
  • the reaction part 130 may have multiple reaction chambers (as shown in the figure, two reaction chambers are shown: a first reaction chamber 131 and a second reaction chamber 132), and different types of reaction chambers are to be tested.
  • the samples can be separately provided in different reaction chambers and mixed with the corresponding reaction reagents.
  • the first reaction chamber and the second reaction chamber in the reaction part 130 may be one reaction chamber, and the sample to be tested is sequentially supplied into the reaction chamber and mixed with the corresponding reaction reagents and then supplied to the detection part. 140 for testing, or adding to the mixed solution in a reaction chamber is for mixing.
  • the specific structure of the reaction part 130 can be determined according to actual needs.
  • the first suspension and the second suspension are respectively supplied to the electrical impedance detection part 141 and the optical detection part 142 of the detection part 140 in this embodiment.
  • the electrical impedance detection part 141 is conventionally equipped with a DC impedance detector and a flow path of a non-focused flow hole or a focused flow hole.
  • a DC impedance detector When particles or blood cells suspended in the conductive solution pass through small holes provided in the flow path, electrical signals can be measured based on changes in impedance.
  • the pulse shape, height, and width of the impedance signal are directly related to the size or volume of the particle, and can be converted into the volume of the particle.
  • the frequency histogram of the impedance signal can reflect the size distribution of these particles (for example, in the form of a histogram).
  • the optical detection part 142 may conventionally include a light source, an optical flow chamber, and at least one optical detector.
  • the optical flow cell is a focused-flow cell suitable for detecting light scattering signals and fluorescent signals.
  • optical flow chambers used in existing flow cytometers and blood analyzers can be used in the blood analysis system of the present invention.
  • the incident light beam from the light source irradiates the particle passing through the detection hole, thereby scattering in all directions.
  • fluorescence can also be emitted. Therefore, optical detectors can be arranged at different angles relative to the incident light beam to capture the scattered light and/or fluorescence.
  • the detector may be arranged at an angle of about 1° to about 10° with respect to the incident light beam to capture forward light scattered signals.
  • the forward light scattering signal may be collected at an angle of about 2° to about 6° in the incident beam.
  • the optical detector is arranged at an angle of about 90° with respect to the incident beam to capture the signal scattered by the side light.
  • the fluorescence detector can also be arranged at an angle of about 90° with respect to the incident beam.
  • the optical detector that captures the lateral light scattering signal may also be arranged at a high angle of about 65° to about 115° with respect to the incident light beam.
  • the electrical impedance detection unit 141 and the optical detection unit 142 that can be used in this embodiment of the present invention can be any suitable known devices, and therefore will not be repeated here.
  • the blood analysis system of the present invention also has a controller 150.
  • the controller 150 is coupled to the sampling part, the reagent supply part, the reaction part, and the detection system, and controls the actions of the sampling part, the reagent supply part, the reaction part, and the detection system.
  • the blood analysis system of the present invention also has a processor 160.
  • the processor can execute a computer program stored in a memory (not shown) to obtain corrected red blood cell volume distribution information (histogram) in the aforementioned blood sample analysis method, and further obtain red blood cell count and other morphology. The results, as well as the analysis of other particles in the blood and obtain relevant results.
  • the blood analysis system of the present invention may also have a user interface (not shown).
  • the user interface may include an input device and an output device.
  • the output device is used to output the detection results obtained by the method according to the present invention, including graphics and data.
  • the output device can also output the normal value reference range of each parameter at the same time, and indicate abnormal conditions.
  • the output test results can also be marked if there is a certain difference between the corrected test results and the pre-corrected test results.
  • the blood analysis system of the present invention may also have a storage device (not shown). According to other embodiments, the storage device may also be connected to the blood analysis system of the present invention through an external device.
  • the storage device may store basic programs and data structures used to implement the functions of various aspects of the methods described herein.
  • a storage device may include one or more memories and one or more non-transitory computer-readable storage media. Non-transitory computer-readable storage media may include hard drives, floppy disks, optical disks, secure digital memory cards (SD cards), flash memory, and the like.
  • the memory may include a main random access memory (RAM) or dynamic RAM (DRAM) for storing program treatments and data, and a read-only memory (ROM) for storing fixed instructions.
  • RAM main random access memory
  • DRAM dynamic RAM
  • ROM read-only memory
  • the non-transitory computer readable medium is programmed by a computer application program to realize the functions disclosed herein, and the corresponding program is executed by one or more processors.
  • the processor executes the computer application stored in the non-transitory computer-readable medium, the processor corrects the first volume distribution information according to the method disclosed herein and further obtains the red blood cell count and related morphological parameters.
  • FIG. 1B shows a schematic diagram of the step flow of the second embodiment
  • Fig. 2B shows a schematic structural diagram of the blood analysis system used in this embodiment.
  • the second embodiment differs from the first embodiment described above mainly in that the second suspension obtained after the second sample is mixed with the second reagent containing the hemolytic agent (step S42) is also subjected to electrical impedance detection (step S43), Thereby, a third histogram about white blood cells can be obtained based on the second threshold (step S44).
  • the corresponding blood analysis system 200 used in this embodiment may have a sampling part 210, a reagent supply part 220 (shown with a reservoir A221 and a reservoir B222), and a reaction part 230 (shown in the figure) which are substantially the same as the blood analysis system 100. It shows a first reaction chamber 231 and a second reaction chamber 232) and a controller 250. The difference is that the detection part 240 may only include the electrical impedance detection part 241.
  • the second electrical signal obtained in step S43 may be inconsistent with the white blood cell volume value obtained from the first electrical signal obtained in step 23.
  • the inventor found through research that the trend of cell volume change is related to the hemolytic agent used.
  • the volume adjustment coefficient K can be obtained through statistics, and the white blood cell volume obtained by the first electrical signal analysis and the white blood cell volume obtained by the second electrical signal analysis can be obtained.
  • VOL2 K ⁇ VOL1 (3).
  • a sample containing only white blood cells can be used as the first test sample and the second test sample to perform the same processing and detection as in this embodiment. Compare the volumes corresponding to the peaks of the two histograms, or calculate and compare the average cell volumes of the two to obtain the value of K.
  • the volume distribution obtained by the second electrical signal is corrected according to the K value, and the volume distribution information of the whole particle event (that is, the third volume distribution information, such as the third histogram) is obtained.
  • the coefficient K’ can also be obtained directly based on the ratio of the white blood cell electrical signal (such as the average electrical signal) obtained by the non-hemolysis channel and the hemolysis channel:
  • the second electrical signal can be directly corrected, and the third histogram can be obtained from the corrected second electrical signal.
  • step S17 the corrected red blood cell volume distribution histogram is obtained by correcting the first histogram with the third histogram, and further, the red blood cell count and other morphological distribution results are obtained in step S18.
  • the processors 160, 260 can perform the above analysis steps to obtain the first histogram and the third histogram respectively, and perform correction to obtain the corrected volume distribution histogram of red blood cells, thereby further obtaining the red blood cell count And other morphological distribution results.
  • the present invention further provides a computer-readable storage medium.
  • a computer program is stored on the computer-readable storage medium, and when the computer program is executed by the above-mentioned processor, the steps of the above-mentioned blood sample analysis method are realized.
  • the computer-readable storage medium may be a memory such as FRAM, ROM, PROM, EPROM, EEPROM, Flash Memory, magnetic surface memory, optical disk, or CD-ROM.
  • the BC-6800 blood analyzer includes a whole blood cell technology (CBC) module and a classification module.
  • the CBC module includes a first mixing chamber and a DC impedance detector.
  • the mixing chamber is configured to mix a sample taken from the blood sample with the diluent to form a first test solution
  • the DC impedance detector is configured to measure the flow through the small hole provided in the circulation path.
  • the classification module includes a second mixing chamber, an infrared semiconductor laser and multiple optical detectors.
  • the mixing chamber is configured to mix another sample taken from the same blood sample with a hemolytic agent and optional fluorescent dye to form a second test solution.
  • the infrared semiconductor laser is used as a light source to emit a light beam aimed at the detection hole of the optical flow chamber.
  • the plurality of optical detectors includes a forward scattered light detector capable of detecting forward light scattering signals emitted from the detection hole and at an angle of about 1° to about 10° with the incident light beam of the light source, and capable of detecting A side-scattered light detector that emits a side-scattered signal at an angle of about 65° to about 115° with the incident beam from the detection hole, and a fluorescence detector that can detect the fluorescent signal.
  • Other existing blood analyzers are also suitable for the solution disclosed in this application, as long as these detectors can detect forward scattered light, side scattered light and/or fluorescence signals. It can also detect light scattering signals at low and middle angles from about 8° to about 24° to the incident beam, or detect light scattering signals at high and middle angles from about 25° to about 65° to the incident beam, instead of the aforementioned side scattered light. , Combined with other light signals, can distinguish white blood cells from other cells, and then obtain a white blood cell volume distribution histogram based on the forward scattered light of white blood cells.
  • Example 1 Using BASO channel to analyze red blood cells in a sample
  • the CBC module 4 ⁇ L of anticoagulated whole blood sample was mixed with 1.5mL M-68DS diluent (Shenzhen Mindray Biomedical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the first histogram is obtained through analysis (the particles contained therein are denoted as particle group 1, see Figure 3).
  • the classification module 20 ⁇ L of anticoagulated whole blood sample taken from the same blood sample is mixed with 1 mL M-68LD Lyse to dissolve red blood cells to form a second test solution.
  • the M-68LD Lyse is an aqueous solution containing cationic surfactants, non-ionic surfactants and anionic compounds, used to dissolve red blood cells in blood samples.
  • the second test solution is transported to the optical detection system, the scattered light detector detects the forward scattered light and the side scattered light, and analyzes the light intensity of the obtained forward and side scattered light, and compares the particle group of white blood cells and other cells. Distinguish (denoted as particle swarm 2), as shown in the scatter plot in Figure 4.
  • the forward scattered light intensity of each cell in the white blood cell group is calculated using the aforementioned formula (1) to obtain the derived volume of each cell, thereby obtaining the second histogram.
  • the histogram of particle swarm 2 is subtracted from the histogram of particle swarm 1 to obtain a corrected histogram of red blood cell particle swarm (see FIG. 6).
  • Example 2 Using BASO channel to analyze red blood cells in a sample
  • Example 7 Another blood sample was tested in the same way as in Example 1. First, the first test solution is detected in the CBC module to obtain the first histogram (the particles contained therein are denoted as particle group 1, see Fig. 7).
  • the second test solution is detected in the classification module to obtain forward scattered light and side scattered light, and the light intensity of the obtained forward and side scattered light is analyzed, and the white blood cell particle group is distinguished from other cells ( Denoted as particle swarm 2), as shown in the scatter plot in Figure 8.
  • the forward scattered light intensity of each cell in the white blood cell group is calculated using the aforementioned formula (1) to obtain the derived volume of each cell, thereby obtaining the second histogram.
  • the first histogram and the second histogram are overlapped, as shown in FIG. 9A, where the enlarged view of the 100-300fL region (see FIG. 9B) can clearly see the curve of the particle group 2 below the curve of the particle group 1.
  • Example 3 Using DIFF channel to analyze red blood cells in a sample
  • CBC module 4 ⁇ L of anticoagulated whole blood sample taken from a blood sample is mixed with 1.5mL M-68DS diluent (Shenzhen Mindray Biomedical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the red blood cell area is distinguished according to the threshold value preset by the system (the particles contained in it are marked as particle group 1, see Figure 11), and the first one is obtained.
  • Square graph 4 ⁇ L of anticoagulated whole blood sample taken from a blood sample is mixed with 1.5mL M-68DS diluent (Shenzhen Mindray Biomedical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the red blood cell area is distinguished according to the threshold value preset by the system (the particles contained in it are marked as particle group 1, see Figure 11), and the first one is obtained.
  • the classification module 20 ⁇ L of anticoagulated whole blood sample taken from the same blood sample is mixed with 1mL M-68LD Lyse and 20 ⁇ LM-68FD dyes (both Shenzhen Mindray Biomedical Electronics Co., Ltd. products) to dissolve red blood cells and have nucleic acid substances Stained blood cells to form a second test solution.
  • the second test solution is transported to the optical detection system, where a scattered light detector detects scattered light and a fluorescence detector detects fluorescence.
  • the obtained side-scattered light and fluorescent light intensity are analyzed, and the particle group of white blood cells is distinguished from other cells (denoted as particle group 2), as shown in the scatter diagram of FIG. 12.
  • the forward scattered light intensity of each cell in the white blood cell group is calculated using the aforementioned formula (1) to obtain the derived volume of each cell, thereby obtaining the second histogram.
  • the histogram of particle swarm 2 is subtracted from the histogram of particle swarm 1 to obtain a corrected histogram of red blood cell particle swarm (see FIG. 14).
  • Example 4 Using the NRBC channel to analyze the red blood cells in the sample
  • CBC module 4 ⁇ L of anticoagulated whole blood sample taken from a blood sample is mixed with 1.5 mL M-68DS diluent (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the red blood cell area is distinguished according to the threshold value preset by the system (the particles contained in it are marked as particle group 1, see Figure 15), and the first one is obtained.
  • Square graph 4 ⁇ L of anticoagulated whole blood sample taken from a blood sample is mixed with 1.5 mL M-68DS diluent (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the red blood cell area is distinguished according to the threshold value preset by the system (the particles contained in it are marked as particle group 1, see Figure 15), and the first one
  • the classification module 20 ⁇ L of anticoagulated whole blood sample taken from the same blood sample is mixed with 1mL M-68LD Lyse and 20 ⁇ LM-68FD dyes (both Shenzhen Mindray Biomedical Electronics Co., Ltd. products) to dissolve red blood cells and have nucleic acid substances Stained blood cells to form a second test solution.
  • the second test solution is transported to the optical detection system, the scattered light is detected by the scattered light detector, and the fluorescence is detected by the fluorescence detector.
  • the light intensity of the obtained forward scattered light and fluorescence is analyzed to distinguish the particle group of white blood cells from other cells (denoted as particle group 2), as shown in the scatter diagram of FIG. 16.
  • the forward scattered light intensity of each cell in the white blood cell population is calculated using the aforementioned formula (1) to obtain the derived volume of each cell, thereby obtaining the second histogram.
  • the histogram of the particle swarm 2 is subtracted from the histogram of the particle swarm 1, and a corrected histogram of the red blood cell particle group is obtained (see FIG. 18).
  • Example 5 Analyze the red blood cells in the sample using the electrical impedance method
  • the CBC module 4 ⁇ L of anticoagulated whole blood sample was mixed with 1.5mL M-68DS diluent (Shenzhen Mindray Biomedical Electronics Co., Ltd.) to form the first test solution.
  • the first test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and the first histogram is obtained after analysis (the particles contained therein are denoted as particle group 1, see Figure 19).
  • the M-68LD Lyse is an aqueous solution containing cationic surfactants, nonionic surfactants and anionic compounds.
  • the second test solution is delivered to the DC impedance detector, the impedance electrical signal is detected, and a second histogram is obtained after analysis (the particles contained therein are denoted as particle group 2, see Figure 20).
  • the histogram of particle swarm 2 is subtracted from the histogram of particle swarm 1 to obtain a corrected histogram of red blood cell particle swarm (see FIG. 22).
  • the second test solution contains a hemolytic agent
  • the cells will shrink to a certain extent and their volume will change.
  • This causes the white blood cell volume of the particle group 2 to be inconsistent with the white blood cell volume of the particle group 1.
  • the study found that the trend of cell volume change is related to the hemolytic agent used.
  • a sample containing only white blood cells can be used as the first test sample and the second test sample for detection. Compare the volume corresponding to the peak of the two histograms, or calculate the average cell volume of the two to obtain the value of K.
  • Example 2 Take the same blood sample as in Example 1, add it to the compacted tube and centrifuge at low speed to remove the white blood cell layer and the plasma layer. The centrifuged sample is then supplemented with diluent to the initial volume. The histogram of the red blood cell particle group obtained directly was measured by the electrical impedance method on the same equipment as in Example 1, as shown in FIG. 23.
  • Example 1 the red blood cell counts and related parameters obtained in Example 1 and Comparative Example 1 are shown in Table 1 below, where the white blood cell content in the sample was detected to be 291.6 ⁇ 10 ⁇ 9/L. It can be seen from Table 1 that the red blood cell counts and various morphological parameters obtained by the two methods are consistent with each other.

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Abstract

分析血液样本中红细胞的方法及血液分析系统(100)。该方法包括：从该血液样本获取第一试样，使该第一试样与第一反应试剂混合得到第一混悬液，该第一混悬液中，红细胞基本维持原有形态；从该血液样本获取第二试样，使该第二试样与含有溶血剂的第二反应试剂混合得到第二混悬液，该第二混悬液中，红细胞被裂解；检测该第一混悬液，获得逐个细胞的第一电信号，并根据该第一电信号获得细胞的第一体积分布信息；检测该第二混悬液，获得逐个细胞的光学信息，通过该光学信息识别白细胞，至少根据该白细胞的前向散射光信息获得白细胞的第二体积分布信息，或者检测该第二混悬液，获得逐个细胞的第二电信号，并根据该第二电信号获得白细胞的第三体积分布信息；用该第二体积分布信息或该第三体积分布信息校正该第一体积分布信息，获得该血液样本中红细胞的校正体积分布信息。

Description

分析血液样本中红细胞方法及血液分析系统 技术领域
本发明涉及血细胞检测领域，具体涉及红细胞分析方法及其分析系统。
背景技术
血细胞分析仪是一种可检测血液中细胞相关信息的仪器，可以对白细胞(WBC)、红细胞(RBC)、血小板(PLT)等细胞进行计数及分类。
对于红细胞和血小板的计数，最常采用的方法有诸如小孔电阻抗法。在该方法中，当血液中的细胞粒子通过小孔时，引起瞬间电压变化，形成电压脉冲。电压脉冲的强度反映了细胞的体积大小，其数量反映了通过微孔的细胞数量，由此获得所测血细胞的体积分布直方图，通过直方图，可以区分血小板和红细胞。
采用小孔阻抗法的血液细胞分析仪除了能获得红细胞的计数值，还能通过红细胞的直方图分布参数，获得常用的红细胞参数，如平均红细胞体积(MCV，Mean Corpuscular Volume)、平均红细胞血红蛋白含量(MCH，Mean Corpuscular Hemoglobin)、平均红细胞血红蛋白浓度(MCHC，Mean Corpuscular Hemoglobin Concentration)、红细胞分布宽度变异系数(RDW-CV，Red Blood Cell Distribution Width Coefficient of Variation)、红细胞分布宽度标准差(RDW-SD，Red Blood Cell Distribution Width Standard Deviation)、红细胞压积(HCT，Hematocrit)等。
然而，实际上，红细胞中可能存在较大体积的红细胞，而一些白细胞如小淋巴细胞，其体积也可能较小，导致二者在直方图上无法良好区分。
在正常情况下，血液中白细胞的数量远远低于红细胞，二者数量相差约1000倍。因此，少量白细胞对红细胞的计数、血红蛋白含量HGB、MCV等参数测定的影响几乎可以忽略。然而，已有报道，当白细胞超过40.0×10^9/L时，白细胞会对血红蛋白含量的分析产生影响，也会使红细胞计数、MCV等形态参数假性增高。最终导致红细胞计数值和相关参数计算结果不准确。
为获得较为准确的红细胞参数可将全血样本加入压积管低速离心，去除白细胞层及血浆层，然后加入等量稀释液混匀后再用分析仪进行检测。这显然增加了检测的复杂度，不能满足对血液进行快速分析的要求。
此外，曾素根等(检验医学,2010,25(3):244-246)也报道了某些疾病(如白血病)引起白血病异常增高会干扰所有型号的血液分析仪的红细胞参数，并给出了一种简单的校正方法。该方法是直接用原始红细胞计数减去白细胞的计数，从而获得校正的红细胞计数。然而，该方法只能获得红细胞的计数，而不能获得红细胞的全部形态参数，实际应用范围过窄。
因此亟需一种能够快速且准确获取RBC计数值和形态参数的方法及分析仪。
发明内容
针对目前以电阻抗法原理对红细胞进行测量的血液细胞分析仪，在检测高值白细胞细胞样本时，其红细胞计数结果及形态参数测量结果易被干扰的缺陷，提供一种去除白细胞对红细胞干扰的分析方法以及相应的血细胞分析仪。
根据本发明的第一方面，提供一种分析样本中红细胞的方法。所述方法包括以下步 骤：
从所述血液样本获取第一试样，使所述第一试样与第一反应试剂混合得到第一混悬液，其中所述第一混悬液中，红细胞基本维持原有形态；
从所述血液样本获取第二试样，使所述第二试样与含有溶血剂的第二反应试剂混合得到第二混悬液，其中所述第二混悬液中，红细胞被裂解；
检测所述第一混悬液，获得逐个细胞的第一电信号，并根据所述第一电信号获得细胞的第一体积分布信息；
检测所述第二混悬液，获得逐个细胞的光学信息，通过所述光学信息识别白细胞，至少根据所述白细胞的前向散射光信息获得白细胞的第二体积分布信息，或者
检测所述第二混悬液，获得逐个细胞的第二电信号，并根据所述第二电信号获得白细胞的第三体积分布信息；
用所述第二体积分布信息或所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
根据本发明的第二方面，提供一种血液分析系统。所述血液分析系统包括：采样部、试剂供应部、反应部、检测部、控制器和处理器，其中：
所述采样部，配置为从血液样本获取至少两份试样，并将所述试样分别输送到所述反应部；
所述试剂供应部，配置为将所需的反应试剂供应到所述反应部；
所述反应部，包括第一反应室，用于将第一试样与第一反应试剂混合制备第一混悬液；和第二反应室，用于将第二试样与第二反应试剂混合制备第二混悬液，且所述反应部配置为将第一混悬液和第二混悬液分别供应到所述检测部；
所述检测部，包括电学检测部和光学检测部，其中所述电学检测部配置为使第一混悬液中粒子逐个通过电阻检测器，以获得第一混悬液中粒子的第一电信号，和所述光学检测部配置为使第二混悬液中的粒子逐个通过光学检测器，以获得第二混悬液中粒子的光学信息；
所述控制器，所述控制器和所述采样部、试剂供应部、反应部和检测部偶联，控制所述采样部、试剂供应部、反应部和检测部的动作；和
所述处理器，所述处理器和检测部耦合，其中
所述处理器从所述检测部获取所述电信号和所述光学信息，根据所述电信号获得第一体积分布信息，根据所述光学信息获得白细胞分布信息，并利用前向散射光强度获得第二体积分布信息，和用所述第二体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
根据本发明的第三方面，提供又一种血液分析系统。所述血液分析系统包括：
采样部、试剂供应部、反应部、检测部和控制器，其中：
采样部，配置为用于从血液样本获取至少两份试样，并将所述试样分别输送到所述反应部；
试剂供应部，配置为将所需的反应试剂供应到所述反应部；
反应部，包括第一反应室，用于将第一试样与第一反应试剂混合制备第一混悬液，和第二反应室，用于将第二试样与第二反应试剂混合制备第二混悬液，且所述反应部配置为将第一混悬液和第二混悬液分别供应到所述检测部；
检测部，包括电学检测部，其中所述电学检测部配置为使所述混悬液中粒子逐个通过电阻检测器，以获得第一混悬液的第一电信号，和第二混悬液的第二电信号；
控制器，所述控制器和所述采样部、试剂供应部、反应部和检测部偶联，控制所述采样部、试剂供应部、反应部和检测部的动作；和
处理器，所述处理器和检测部耦合，其中
所述处理器从所述检测部获取所述第一和第二电信号，根据所述第一电信号获得第一体积分布信息，根据所述第二电信号获得第三体积分布信息，和用所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本的红细胞的校正体积分布信息。
根据本发明的第四方面，提供一种计算机可读存储介质，所述计算机可读存储介质上存储有计算机程序，其特征在于，该计算机程序被处理器执行时实现以下步骤：
获取通过检测第一混悬液获得的第一电信号，并根据所述第一电信号获得第一体积分布信息，其中所述第一混悬液由从血液样本获取的第一试样与第一反应试剂混合得到，且所述电信号通过使所述第一混悬液通过电学检测部的检测得到；
获取通过检测第二混悬液获得的光学信息，并通过所述光学信息获得白细胞分布信息，并利用前向散射光强度获得第二体积分布信息，其中所述第二混悬液由从所述血液样本获取的第二试样与至少含有溶血剂的第二反应试剂混合得到，所述光学信息通过使所述第二混悬液通过光学检测部的检测得到，
或者
获取通过检测第二混悬液获得的第二电信号，并根据所述第二电信号获得第三体积分布信息，其中所述第二混悬液由从血液样本获取的第二试样与至少含有溶血剂的第二反应试剂混合得到，且所述电信号通过使所述第二混悬液通过所述电学检测部的检测得到；和
用所述第二体积分布信息或所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
本发明的血细胞分析方法利用现有白细胞(溶血)光学检测通道，或者仅利用白细胞(溶血)电阻抗检测通道，就能够有效排除样本中，特别是白细胞含量较高的样本中，白细胞对红细胞分析的干扰，以获得准确的红细胞计数及相关参数，从而为临床诊断和治疗提供准确的检验信息。常规的血液分析仪中通常可以在WBC/BASO通道、DIFF通道、NRBC通道、电阻抗检测通道检测白细胞，获得计数值。通过以下详述的实施例证实，无论用哪种通道获得的白细胞体积分布信息来校正电阻抗法获得的包含白细胞和红细胞的体积分布信息，均可获得准确的红细胞体积分布信息。此外，在白细胞(溶血)电阻抗检测通道中，通过对经溶血的样本的检测，也可获得白细胞体积分布信息，用来校正包含白细胞和红细胞的体积分布信息，获得准确的红细胞体积分布信息。本发明的方法和分析系统能够以简单的方法获得准确的红细胞计数及相关形态参数。
附图说明
图1A是根据本发明的第一实施方式对血液样本中的红细胞进行分析的方法步骤的流程示意图；
图1B是根据本发明的第二实施方式对血液样本中的红细胞进行分析的方法步骤的流程示意图；
图2A是根据本发明的第一实施方式血液分析系统的结构示意框图；
图2B是根据本发明的第儿实施方式血液分析系统的结构示意框图；
图3是根据实施例1的第一试液的第一直方图；
图4是根据实施例1的第二试液的白细胞粒子群的散点图；
图5是根据实施例1的第一直方图和第二直方图的重叠图，其中A为全图，B为100-300fL区域的放大图；
图6是根据实施例1的经校正的红细胞直方图；
图7是根据实施例2的第一试液的第一直方图；
图8是根据实施例2的第二试液的白细胞粒子群的散点图；
图9是根据实施例2的第一直方图和第二直方图的重叠图，其中A为全图，B为100-300fL区域的放大图；
图10是根据实施例2的经校正的红细胞直方图；
图11是根据实施例3的第一试液的第一直方图；
图12是根据实施例3的第二试液的白细胞粒子群的散点图；
图13是根据实施例3的第一直方图和第二直方图的重叠图，其中A为全图，B为100-300fL区域的放大图；
图14是根据实施例3的经校正的红细胞直方图；
图15是根据实施例4的第一试液的第一直方图；
图16是根据实施例4的第二试液的白细胞粒子群的散点图；
图17是根据实施例4的第一直方图和第二直方图的重叠图，其中A为全图，B为150-300fL区域的放大图；
图18是根据实施例4的经校正的红细胞直方图；
图19是根据实施例5的第一试液的第一直方图；
图20是根据实施例5的第二试液的白细胞粒子群的第二直方图；
图21是根据实施例5的第一直方图和第二直方图的重叠图，其中A为全图，B为100-200fL区域的放大图；
图22是根据实施例5的经校正的红细胞直方图；和
图23是根据对比例1直接测试获得的红细胞的直方图。
具体实施方式
下面将结合附图，对本发明的具体实施方式进行清楚、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做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，本文定义优先。
本上下文中提及的术语“直方图”是细胞体积分布的图形形式，是呈现连续变量概率分布的常见形式。可选地，该细胞体积分布也可以采用与该体积直方图具有等同或相近分辨率的表格或列表的数字形式呈现，或者采用任何本领域已知的其他适合的方式呈现。
针对当前血液分析仪针对高白细胞样本的分析存在对红细胞计数及形态参数产生干扰的问题，本发明提供一种简便的去除白细胞干扰的红细胞分析方法。
对于正常样本，白细胞的含量仅为红细胞的千分之一，且白细胞的体积通常比红细胞大。在常规的阻抗法获得的直方图中，白细胞的影响可以忽略不计。但是，对于白细胞含量高的异常样本，用常规的阻抗法获得的直方图中在红细胞的体积范围内混杂了数量较为可观的白细胞粒子，从而影响了红细胞的计数及其他形态学分析结果，并进一步对正确的诊断造成不利影响。
本发明总的来说，利用血液分析仪中已有的通道和检测设备，分析得到白细胞的直方图，并根据白细胞的直方图来校正按照常规的阻抗法检测得到的红细胞直方图，从而获得与真实的红细胞信息相近的校正的直方图，并进一步获得正确的红细胞的计数及其他形态学分析结果。
根据本发明的第一实施方式的分析血液样本中红细胞的方法，包括以下步骤：
从所述血液样本获取第一试样，使所述第一试样与第一反应试剂混合得到第一混悬液，和从所述血液样本获取第二试样，使所述第二试样与含有溶血剂的第二反应试剂混合得到第二混悬液，其中所述第一混悬液中，红细胞基本维持原有形态，而所述第二混悬液中，红细胞被裂解；
检测所述第一混悬液，获得逐个细胞的第一电信号，并根据所述第一电信号获得第一体积分布信息；
检测所述第二混悬液，获得逐个细胞的光学信息，通过所述光学信息获得白细胞分布信息，并至少根据所述白细胞的前向散射光信息获得第二体积分布信息；
用所述第二体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
参见图1A，其中示例了该实施方式的方法流程示意图。在该实施方式中，首先提供血液样本S11。本发明的血液样本通常为全血样本。可以是来自哺乳动物，特别是灵长类动物，更具体地为人类的全血样本。
从所述血液样本获取第一试样与第一反应试剂混合S22，以获得第一混悬液。
所述第一反应试剂通常为稀释剂。本发明对稀释剂没有特别限制，任何适宜用于电阻抗检测的稀释剂均可用于本发明。稀释剂通常包括一种或多种盐，例如碱金属盐，并被调节为等渗的(isotonic)以维持红细胞体积。本发明对稀释剂没有特别限制，任何适宜用于电阻抗检测的稀释剂均可用于本发明。例如可以采用任何适宜的商业血液稀释液，如，深圳迈瑞生物医疗电子股份有限公司(深圳，中国)的M-68DS稀释液、M-53D稀释液等。
经稀释获得的第一混悬液，即第一试液进一步进行电阻抗检测S23，获得第一试样中血细胞的电信号。电阻抗法可为，例如小孔阻抗法、鞘流阻抗法等。
首先获取该第一试样中全粒子事件的电学信息，并进一步由此获得血细胞的体积分布信息(称为第一直方图)S24。
在其他实施方式中，可以区分红细胞和血小板，将红细胞直方图作为第一直方图。同时地，或先后地，从所述血液样本获取第二试样与第二反应试剂混合S32，以获得第二混悬液。
在一种替代方案中，第一试样可顺序进行电阻抗检测和光学检测。在这种情况下，第二试样是经过电阻抗检测后的第一试样进一步与第二检测试剂混合得到。即，第一血液样本与第一检测试剂混合获得第一试样，第一试样经电阻抗检测后进一步与第二检测试剂混合获得第二试样。
该替代方案的好处在于不需要额外的样本量，因而不会给受试者，特别是那些存在取血困难的受试者，带来额外的负担。
本发明的第二反应试剂含有溶血剂，以溶解第二试样中的红细胞。溶血剂可以是阳离子、非离子、阴离子、两亲性表面活性剂中的任意一种或几种的组合。本发明中用于溶解第二试样中红细胞的溶血剂没有特别限制。因此，可以使用任意一种适宜用于血液分析仪白细胞分类的溶解试剂。用于血液分析仪白细胞分类的溶解试剂通常是含有一种或多种上述溶血剂的水溶液。
在一些实施方式中，处理所述第二试样的第二反应试剂中除了含有溶血剂外，还进一步含有用于染色有核血细胞的荧光染料，从而可以通过检测散射光和荧光的强度进一步对白细胞进行分离和/或区分出有核血细胞。例如，可以采用美国专利U.S.8,367,358所描述的溶解试剂配方，其全部公开内容通过引证结合于此。在美国专利U.S.8,367,358中所披露的溶解试剂包括一种阳离子花菁化合物(一种荧光染料)、一种阳离子表面活 性剂、一种非离子表面活性剂和一种阴离子化合物，该溶解试剂可以用于溶解红细胞和使用荧光和光散射测量将白细胞分类为其亚群。其他适宜的荧光染料也可以被用在溶解试剂中。例如，美国专利U.S.8,273,329中所描述的荧光染料，其全部公开内容通过引证结合于此。
此外，在一些实施方式中，荧光染料可以包含在独立的染色溶液中，作为第三反应试剂与不含有荧光染料的作为溶解试剂的第二反应试剂一起使用。所述染色溶液可以在溶解试剂之前、之后或同时加入血液样本以染色有核血细胞。
仍参考图1A，步骤S33中，第二混悬液进一步进行光学检测，从而获得第二混悬液中血细胞的光学信息。
步骤S34中由光学信息可以得到第二混悬液中细胞的光学信息；接着根据粒子的光学特性区分出白细胞粒子群，并进一步根据白细胞的前向散射光强度(其能够反映出细胞体积信息)获得白细胞体积的直方图，作为第二直方图。
由光学信息获得散点图以便识别出白细胞粒子群可采用任何常规的方法。通常通过两种光学信息，诸如前向和侧向散射光的光强度、前向散射光和荧光的光强度、侧向散射光和荧光的光强度等等来获得二维散点图。有必要时，也可以通过三种信息获得三维散点图。本发明对此并没有特别限制。
识别出白细胞粒子群后，可根据白细胞的前向散射光强度通过分析得到白细胞的体积分布信息，并以诸如直方图形式表示。
将光学信息转化为直方图的方法没有特别限制，可以是现有的任何适宜方法，例如，依据Mie散射理论利用前向光散射信号通过计算获得粒子的一维体积分布，即直方图。
此外，本发明中的散点图的直方化还可以采用下述任意一种方法。
一种方法是利用粒子的前向光散射信息，根据以下公式(1)计算每个粒子的衍生体积Vol f1。
Vol f1＝α×FSC   (1)
其中FSC为二维散点图中待转换粒子群(本发明中具体为单个白细胞)中的一个单独事件(individual event)的前向散射光的强度，α为常数，其取值与检测设备相关，不同的检测设备α值可不同。
另一种方法是利用粒子的前向光散射信息，根据公式(2)计算每个粒子的衍生体积Vol f2。
Vol f2＝β×exp(γ·FSC)    (2)
其中FSC同样为二维散点图中待转换粒子群(本发明中具体为单个白细胞)中的一个单独事件(individual event)的前向散射光的强度，β和γ为常数。同样的，其取值与检测设备相关，不同的检测设备β和γ值可不同。
通过将第二粒子群的每个粒子的前向散射光的光强度转化为衍生体积Vol  f1或者Vol f2，能够得到与DC阻抗法获得的直方图中的白细胞体积相关的体积分布直方图。
本发明人研究发现，通过将光学检测得到的白细胞散点图直方化能够很好地匹配由电阻抗法检测得到的直方图。因此，本发明得以利用光学检测得到的白细胞信息来校正电阻抗法检测得到的红细胞和白细胞的混合信息，并进而获得准确的红细胞信息。而且，经验证，在该实施方式中，不论采用哪个通道(诸如WBC/BASO、DIFF或NRBC)进行白细胞检测所获得的白细胞光学信息，在转化为直方图后，都能用于校正电阻抗法获得的直方图。以下实施例也证实了所获得的经校正的红细胞直方图与事先对血液样本进行处理除去白细胞后再用电阻抗法检测获得的直方图结果一致。
和现有技术中的一些简单的通过扣除白细胞计数的校正法不同，本发明的方法可获得红细胞的体积分布信息，由此可进一步获得相关的红细胞形态分布数据，而并不仅限 于对红细胞计数的校正。
如图1A所示，用第二直方图对第一直方图进行校正(步骤S15)后获得的直方图可获取红细胞计数和其他形态参数(步骤S16)。所述其他形态参数可选自如下组中的至少一个参数：平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度变异系数(RDW-CV)、红细胞分布宽度标准差(RDW-SD)、红细胞压积(HCT)。
此外，本发明的方法没有改变信号的检测原理，可以应用在多种检测场合。即，本发明的方法可利用血液分析仪中的多种溶血后对试样进行检测场景下。因此，在用血细胞分析仪进行血液样本检测时，在获得正确的红细胞相关参数的同时，可获得任何所需的常规通过电阻抗法和/或光学检测可获得的血液样本的其他参数。例如：根据侧向和前向光散射的光强度可区分嗜碱性粒细胞和其他白细胞粒子；根据前向光散射的光强度和荧光强度也可区分嗜碱性粒细胞和其他白细胞粒子，还可识别有核酸物质的血细胞，如有核红细胞；以及根据侧向光散射的光强度和荧光强度对白细胞进行四分类，区分嗜酸性粒细胞、中性粒细胞、单核细胞和淋巴细胞，但不限于此。
在获得相关信息和参数后，本发明的方法还包括输出所需信息和/或参数的步骤。其中，可根据白细胞计数是否异常(例如超过第一阈值)，或者可根据校正前后的红细胞计数的差异是否超出正常范围(例如超过第二阈值)，进一步对输出的经校正后得到的信息(如直方图)和/或参数(如红细胞计数及其他形态参数)进行标示。
根据需要，本发明的方法不仅可以输出经校正的直方图和相关参数，还可输出校正前的第一直方图，甚至叠加了用于校正的第二或第三直方图的第一直方图。
除输出检测结果外，还可进一步输出各个项目的正常范围作为参考。
进一步结合图2A，其中示出了可用于上述方法的血液分析仪100的结构示意图。
血液分析仪100常规地包括采样部110、试剂供应部120、反应部130、检测部140、控制器150和处理器160。当然还可以包括其他必要装置，例如显示部、输出和输入部、壳体以及必要的泵和液路系统等，这些装置在此不再赘述。
采样部110可包括吸液装置、分样器等，以从血液样本中吸取用于检测的第一和第二试样，并把第一试样供给到反应部130的第一反应室131，并把第二试样供给到第二反应室132。
试剂供应部120用于把所需的反应试剂供应到反应部。图2A中根据一种具体实施方式示出了具有两个贮液槽的试剂供应部120：贮液槽A121和贮液槽B122，分别用于贮存如上所述的第一反应试剂和第二反应试剂，并将它们分别供应到反应部130的第一反应室131，并把第二试样供给到第二反应室132，以与其中的第一和第二试样分别混合得到第一混悬液和第二混悬液。试剂供应部120还可具有更多的贮液槽。例如如上所述的对细胞进行荧光染色的实施方式中，试剂贮存部120还可具有第三贮液槽以单独贮存荧光染色液。
在其他实施方式中，试剂供应部也可以没有贮液槽。试剂供应部从机外的试剂桶将所需的反应试剂抽吸并供应到反应部。
根据一种实施方式，所述反应部130可具有多个反应室(如图所示，其中示出了两个反应室：第一反应室131和第二反应室132)，不同的待测试样可分别提供至不同的反应室中与相应的反应试剂进行混合。根据另一种实施方式，反应部130中的第一反应室和第二反应室可以为一个反应室，待测试样顺序提供到该反应室中与相应的反应试剂混合并进而供应到检测部140进行检测，或者在一个反应室中混合液中再加入是进行混合。反应部130的具体结构可根据实际需要而确定。
混合反应完成后，第一混悬液和第二混悬液在该实施方式中分别供应至检测部140 的电阻抗检测部141和光学检测部142。
电阻抗检测部141常规地，装配有DC阻抗检测器和非聚焦流动孔或聚焦流动孔的流动路径。当悬浮在导电溶液中的粒子或血细胞通过设置在流动路径上的小孔时，可以基于阻抗变化测得电信号。该阻抗信号的脉冲形状、高度和宽度与粒子的尺寸或体积直接相关，并可以被转换为粒子的体积。当具有不同尺寸的两种或多种粒子被测量时，由阻抗信号的频率直方图可以反映这些粒子的尺寸分布(例如以直方图的形式体现)。通过配置有DC阻抗测量设备的血液分析仪对血细胞进行检测的方法是已知的，诸如美国专利US2,656,508和US3,810,011中所描述的，其中这两篇专利的全部公开内容通过引用合并于本文中。
光学检测部142可常规地包括光源、光学流动室和至少一个光学检测器。光学流动室为适于检测光散射信号和荧光信号的聚焦液流的流通池(focused-flow cell)。例如现有的流式细胞仪和血液分析仪中所使用的光学流动室均可用于本发明的血液分析系统中。当粒子通过光学流动室的检测孔(orifice)时，来自光源的入射光束照射在正通过该检测孔的该粒子上，从而向各个方向散射。对于经荧光染色剂染色的粒子，还可发射出荧光。因此，可在相对于该入射光束的不同角度上布置光学检测器以捕获所述散射光和/或荧光。由于不同的粒子群具有不同的光散射特性或根据不同的荧光染料而具有不同的荧光特性，因此可以根据这些光学信息来区分不同的粒子群。在一些实施方式中，可在相对于入射光束呈约1°至约10°的角度上布置检测器以捕获前向光散射的信号。在其他一些实施方式中，前向光散射的信号可以在于入射光束呈约2°至约6°的角度上收集。在相对于入射光束呈约90°的角度上布置光学检测器以捕获侧向光散射的信号。荧光检测器也可布置在相对于入射光束呈约90°的角度上。在一些实施方式中，也可在相对于入射光束呈约65°至约115°的高角度布置捕获侧向光散射的信号的光学检测器。
可用于本发明该实施方式的电阻抗检测部141和光学检测部142可为任何适宜的已知装置，因此在此不再赘述。
本发明的血液分析系统还具有控制器150。所述控制器150和所述采样部、试剂供应部、反应部和检测系统偶联，控制所述采样部、试剂供应部、反应部和检测系统的动作。
本发明的血液分析系统还具有处理器160。所述处理器可执行存储在存储器(未示出)中的计算机程序，以实现前述血液样本分析方法中分析获得校正的血红细胞体积分布信息(直方图)，以及进一步获得红细胞计数及其他形态学结果，以及对血液中其他粒子的分析并获得相关结果。
此外，本发明的血液分析系统还可具有用户界面(未示出)。常规地，用户界面可包括输入设备和输出设备。其中所述输出设备用于输出上述根据本发明的方法获得的包括图形和数据形式的检测结果。根据其他实施方式，所述输出设备还可同时输出各个参数的正常值参考范围，并标示出异常的情况。此外，如前所述对于输出的检测结果经过校正后与校正前相比有一定差异的，也可进行标示。
本发明的血液分析系统还可具有存储装置(未示出)。根据其他实施方式，所述存储装置也可以通过外接设备与本发明的血液分析系统连接。所述存储装置可存储用于实现本文所述的方法的各个方面的功能的基础程序和数据结构。常规地，存储装置可包括一个或多个存储器和一个或多个非暂时性计算机可读存储介质。非暂时性计算机可读存储介质可包括硬盘驱动器、软盘、光盘、安全数字记忆卡(SD卡)、闪存等。存储器可包括用于存储程序治疗和数据的主随机存取存储器(RAM)或动态RAM(DRAM)及用于存储固定指令的只读存储器(ROM)。非暂时性计算机可读介质被计算机应用程序编程以实现本文所公开的功能，并由一个或多个处理器执行相应的程序。当所述处理器 执行存储于非暂时性计算机可读介质的计算机应用时，所述处理器根据本文所公开的方法校正第一体积分布信息并进一步获得红细胞计数及相关形态参数。
进一步结合图1B和图2B说明本发明的第二实施方式。其中图1B中示出了该第二实施方式的步骤流程示意图；图2B示出了用于该实施方式的血液分析系统的结构示意图。
该第二实施方式与上述第一实施方式的区别主要在于第二试样与含有溶血剂的第二试剂混合(步骤S42)后得到的第二混悬液也进行电阻抗检测(步骤S43)，从而根据第二阈值可获得关于白细胞的第三直方图(步骤S44)。
相应的用于该实施方式的血液分析系统200可具有与血液分析系统100大体相同的采样部210、试剂供应部220(图示具有贮液槽A221和贮液槽B222)、反应部230(图示具有第一反应室231和第二反应室232)和控制器250，区别在于检测部240可仅包括电阻抗检测部241。
根据该实施方式，在将第二样本中的红细胞溶血后，也通过电阻抗法进行检测。由于溶血剂除了溶解红细胞外，还会使其他血细胞发生一定程度的皱缩，经步骤S43获得的第二电信号与经步骤23获得的第一电信号得到的白细胞体积数值可能不一致。本发明人经研究发现，细胞体积改变的趋势与所用的溶血剂有关，可以通过统计得到体积调整系数K，并获得由第一电信号分析得到的白细胞体积与由第二电信号分析得到的白细胞体积之间存在函数映射关系：
VOL2＝K×VOL1   (3)。
系数K，可以使用仅含有白细胞的样本分别作为第一测试试样和第二测试试样进行与该实施方式相同的处理和检测。比较两个直方图的峰值对应的体积，或者分别计算二者的平均细胞体积并比较，以获得K的值。
根据K值对第二电信号得到的体积分布进行校正，获得全粒子事件的体积分布信息(即第三体积分布信息，例如第三直方图)。
此外，也可直接根据非溶血通道和溶血通道获得的白细胞电信号(如平均电信号)的比值获得系数K’：
S2＝K’×S1   (4)。
根据K’，可直接对第二电信号进行校正，并通过经校正的第二电信号获得第三直方图。
接着在步骤S17中，通过用第三直方图校正第一直方图，获得校正的红细胞体积分布直方图，并进一步在步骤S18中获得红细胞的计数和其他形态分布结果。
当采用该实施方式时，处理器160，260可执行上述分析步骤，分别获得第一直方图和第三直方图，并进行校正以获得红细胞经校正的体积分布直方图，从而进一步获得红细胞计数和其他形态分布结果。
该实施方式涉及的与第一实施方式中相同的步骤、试剂、部件等在此不再赘述。
此外，本发明进一步提供一种计算机可读存储介质。所述计算机可读存储介质上存储有计算机程序，该计算机程序被上述处理器执行时实现前述血液样本分析方法的步骤。所述计算机可读存储介质可以是FRAM、ROM、PROM、EPROM、EEPROM、Flash Memory、磁表面存储器、光盘、或CD-ROM等存储器。
以下通过具体实施例进一步说明本发明及其有益效果。
以下实施例均使用商业血液分析仪BC-6800(深圳迈瑞生物医疗电子股份有限公司，中国深圳)测量全血样本并进行分析。
BC-6800血液分析仪包括全血细胞技术(CBC)模块和分类模块。该CBC模块包括第一混合室和DC阻抗检测器。该混合室被设置用于将取自血液样本的一份试样与 稀释液混合以形成第一试液，该DC阻抗检测器被设置用于测量流过设置在流通路径上的小孔的该第一试液的DC阻抗信号。该分类模块包括第二混合室、红外半导体激光器和多个光学检测器。该混合室被设置用于将取自同一血液样本的另一份试样与溶血剂及可选的荧光染料混合以形成第二试液。该红外半导体激光器作为光源，发射出对准光学流动室的检测孔的光束。多个光学检测器包括能够检测从所述检测孔发出的、与所述光源的入射光束呈约1°至约10°的角度上的前向光散射信号的前向散射光检测器，能够检测从所述检测孔发出的、与入射光束呈约65°至约115°的角度上的侧向光散射信号的侧向散射光检测器，及能够检测荧光信号的荧光检测器。
其他现有的血液分析仪也适用于本申请公开的方案，只要具有这些检测器能检测前向散射光、侧向散射光和/或荧光信号即可。也可以检测与入射光束约8°至约24°的低中角度的光散射信号，或者检测与入射光束约25°至约65°的角度的高中角度光散射信号，以代替前述侧向散射光，并与其他光信号结合，可区分白细胞和其他细胞，然后基于白细胞的前向散射光得到白细胞体积分布直方图。
实施例1：利用BASO通道分析样本中的红细胞
在CBC模块中，4μL抗凝全血样本用1.5mL M-68DS稀释液(深圳迈瑞生物医疗电子股份有限公司)混合，形成第一试液。第一试液被输送到DC阻抗检测器，阻抗电信号被检测，并经分析得到第一直方图(其中所包含的粒子记为粒子群1，参见图3)。
在分类模块中，取自同一血样的20μL抗凝全血样本与1mL M-68LD Lyse混合以溶解红细胞，形成第二试液。该M-68LD Lyse为含有阳离子表面活性剂、非离子表面活性剂和阴离子化合物的水溶液，用于溶解血液样本中的红细胞的。第二试液被输送到光学检测系统，散射光检测器检测前向散射光和侧向散射光，对获得的前向和侧向散射光的光强度进行分析，将白细胞的粒子群和其他细胞区分(记为粒子群2)，如图4的散点图所示。将白细胞群中每个细胞的前向散射光强度，用前述的公式(1)计算得到每个细胞的衍生体积，从而得到第二直方图。
将第一直方图与第二直方图的重叠，如图5A所示，其中100-300fL区域的放大图(参见图5B)可清晰看到粒子群1的曲线下方位粒子群2的曲线。两条曲线并不重合，说明粒子群1的该区域中混杂了红细胞和白细胞。
通过计算，从粒子群1的直方图中扣除粒子群2的直方图，获得经校正的红细胞粒子群的直方图(参见图6)。
实施例2：利用BASO通道分析样本中的红细胞
按照与实施例1相同的方法检测另一血液样本。首先，在CBC模块中进行检测第一试液获得第一直方图(其中所包含的粒子记为粒子群1，参见图7)。
接着在分类模块中对第二试液进行检测，获得前向散射光和侧向散射光，对获得的前向和侧向散射光的光强度进行分析，将白细胞的粒子群和其他细胞区分(记为粒子群2)，如图8的散点图所示。将白细胞群中每个细胞的前向散射光强度，用前述的公式(1)计算得到每个细胞的衍生体积，从而得到第二直方图。
将第一直方图与第二直方图的重叠，如图9A所示，其中100-300fL区域的放大图(参见图9B)可清晰看到粒子群1的曲线下方粒子群2的曲线。
由图9可见，白细胞在阻抗通道出现两个峰。由图可见，两个白细胞群会干扰红细胞粒子群。校正后的红细胞直方图如图10所示。
实施例3：利用DIFF通道分析样本中的红细胞
在CBC模块中，取自一个血样的4μL抗凝全血样本用1.5mL M-68DS稀释液(深 圳迈瑞生物医疗电子股份有限公司)混合，形成第一试液。第一试液被输送到DC阻抗检测器，阻抗电信号被检测，根据系统预设的阈值，区分出红细胞区域(其中所包含的粒子记为粒子群1，参见图11)，得到第一直方图。
在分类模块中，取自同一血样的20μL抗凝全血样本与1mL M-68LD Lyse和20μLM-68FD染料(均为深圳迈瑞生物医疗电子股份有限公司的产品)混合以溶解红细胞并对有核酸物质的血细胞染色，形成第二试液。第二试液被输送到光学检测系统，其中散射光检测器检测散射光，荧光检测器检测荧光。对获得的侧向散射光和荧光的光强度进行分析，将白细胞的粒子群和其他细胞区分(记为粒子群2)，如图12的散点图所示。将白细胞群中每个细胞的前向散射光强度，用前述的公式(1)计算得到每个细胞的衍生体积，从而得到第二直方图。
将第一直方图与第二直方图的重叠，如图13A所示，其中100-300fL区域的放大图(参见图13B)可清晰看到第一直方图的曲线下方第二直方图的曲线。
通过计算，从粒子群1的直方图中扣除粒子群2的直方图，获得经校正的红细胞粒子群的直方图(参见图14)。
实施例4：利用NRBC通道分析样本中的红细胞
在CBC模块中，取自一个血样的4μL抗凝全血样本用1.5mL M-68DS稀释液(深圳迈瑞生物医疗电子股份有限公司)混合，形成第一试液。第一试液被输送到DC阻抗检测器，阻抗电信号被检测，根据系统预设的阈值，区分出红细胞区域(其中所包含的粒子记为粒子群1，参见图15)，得到第一直方图。
在分类模块中，取自同一血样的20μL抗凝全血样本与1mL M-68LD Lyse和20μLM-68FD染料(均为深圳迈瑞生物医疗电子股份有限公司的产品)混合以溶解红细胞并对有核酸物质的血细胞染色，形成第二试液。该第二试液被输送到光学检测系统，由散射光检测器检测散射光，由荧光检测器检测荧光。对获得的前向散射光和荧光的光强度进行分析，以将白细胞的粒子群和其他细胞区分(记为粒子群2)，如图16的散点图所示。将白细胞群中每个细胞的前向散射光强度，用前述的公式(1)计算得到每个细胞的衍生体积，从而得到第二直方图。
将第一直方图与第二直方图的重叠，如图17A所示，其中150-300fL区域的放大图(参见图17B)可清晰看到第一直方图的曲线下方第二直方图的曲线。
通过计算，从粒子群1的直方图中扣除粒子群2的直方图，获得经校正的红细胞粒子群的直方图(参见图18)。
实施例5：利用电阻抗法分析样本中的红细胞
在CBC模块中，4μL抗凝全血样本用1.5mL M-68DS稀释液(深圳迈瑞生物医疗电子股份有限公司)混合，形成第一试液。第一试液被输送到DC阻抗检测器，阻抗电信号被检测，并经分析得到第一直方图(其中所包含的粒子记为粒子群1，参见图19)。
仍在CBC模块中，取同一样本的4μL抗凝全血样本用与1mL M-68LD Lyse混合以溶解红细胞，形成第二试液。该M-68LD Lyse为含有阳离子表面活性剂、非离子表面活性剂和阴离子化合物的水溶液。第二试液被输送到DC阻抗检测器，阻抗电信号被检测，并经分析得到第二直方图，(其中所包含的粒子记为粒子群2，参见图20)。
将第一直方图与第二直方图的重叠，如图21A所示，其中100-200fL区域的放大图(参见图21B)可清晰看到第一直方图的曲线下方第二直方图的曲线。
通过计算，从粒子群1的直方图中扣除粒子群2的直方图，获得经校正的红细胞粒子群的直方图(参见图22)。
由于第二试液中含有溶血剂，细胞会发生一定程度的皱缩，体积改变。这导致粒子群2的白细胞体积通粒子群1中的白细胞体积不一致。研究发现，细胞体积改变的趋势与所用的溶血剂有关，可以通过统计得到体积调整系数K，将该系数用于衍生体积的计算，从而使得第一和第二直方图中白细胞的体积分布具有可比性。即，粒子群2中的白细胞同粒子群1的白细胞体积存在函数映射关系，VOL2＝K×VOL1。
为了获得上述系数K，可以使用仅含有白细胞的样本分别作为第一测试试样和第二测试试样进行检测。比较两个直方图的峰值对应的体积，或者计算二者的平均细胞体积，获得K的值。
对比例1
取与实施例1相同的血液样品，加入压积管低速离心，去除白细胞层及血浆层。然后将离心处理后的样品补充稀释液至初始体积。在与实施例1相同的设备上用电阻抗法测定直接获得的红细胞粒子群的直方图，如图23所示。
经比较，实施例1和对比例1获得的红细胞计数及相关参数如下表1所示，其中，经检测该样本中白细胞含量为291.6×10^9/L。由表1可知，两种方法获得的红细胞计数及各形态参数均相互吻合。
表1红细胞计数和相关参数值
Figure PCTCN2019112766-appb-000001
以上所述仅为用于解释和说明本发明的示例性实施方式及实施例，而非意欲因此限制本发明的范围。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 (30)

  1. 一种分析血液样本中红细胞的方法，所述方法包括以下步骤：
    从所述血液样本获取第一试样，使所述第一试样与第一反应试剂混合得到第一混悬液，其中所述第一混悬液中，红细胞基本维持原有形态；
    从所述血液样本获取第二试样，使所述第二试样与含有溶血剂的第二反应试剂混合得到第二混悬液，其中所述第二混悬液中，红细胞被裂解；
    检测所述第一混悬液，获得逐个细胞的第一电信号，并根据所述第一电信号获得细胞的第一体积分布信息；
    检测所述第二混悬液，获得逐个细胞的光学信息，通过所述光学信息识别白细胞，至少根据所述白细胞的前向散射光信息获得白细胞的第二体积分布信息，或者
    检测所述第二混悬液，获得逐个细胞的第二电信号，并根据所述第二电信号获得白细胞的第三体积分布信息；
    用所述第二体积分布信息或所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
  2. 根据权利要求1所述的方法，其中所述光学信息包括前向散射光强度。
  3. 根据权利要求2所述的方法，其中所述光学信息还包括侧向散射光强度，所述侧向散射光强度选自中角度散射光强度和/或高角度散射光强度。
  4. 根据权利要求3所述的方法，其中所述方法进一步包括：根据所述光学信息区分出嗜碱性白细胞亚群。
  5. 根据权利要求1所述的方法，其中所述第二反应试剂还含有荧光染料，或者使所述第二试样进一步与含有荧光染料的第三反应试剂混合得到所述第二混悬液。
  6. 根据权利要求5所述的方法，其中所述光学信息还包括荧光强度。
  7. 根据权利要求6所述的方法，其中所述方法进一步包括：根据所述光学信息区分出有核红细胞和白细胞粒子群。
  8. 根据权利要求6所述的方法，其中所述方法进一步包括：根据所述光学信息区分出嗜碱性白细胞亚群。
  9. 根据权利要求5所述的方法，其中所述光学信息包括侧向散射光强度和荧光强度。
  10. 根据权利要求9所述的方法，其中所述方法进一步包括：根据所述光学信息区分白细胞亚群为淋巴细胞、单核细胞、中性粒细胞和嗜酸性粒细胞亚群。
  11. 根据权利要求1所述的方法，其中所述方法进一步包括：根据所述第二电信号区分白细胞亚群为淋巴细胞、单核细胞和粒细胞亚群。
  12. 根据权利要求1所述的方法，其中所述第一、第二和第三体积分布信息以及校正的体积分布信息为直方图。
  13. 根据权利要求1所述的方法，其中所述方法还包括输出由所述红细胞的校正体积分布信息获取的选自如下组中的至少一个信息：红细胞计数、平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度变异系数(RDW-CV)、红细胞分布宽度标准差(RDW-SD)和红细胞压积(HCT)；和/或由所述白细胞分布信息获得白细胞总计数和白细胞亚群的计数。
  14. 根据权利要求1所述的方法，其中所述方法还包括输出所述校正体积分布信息，以及可选的第一和第二体积分布信息，或可选的第一和第三体积分布信息。
  15. 根据权利要求13或14所述的方法，其中当白细胞计数超过第一阈值时，或者当校正前后红细胞计数的差值超过第二阈值时，对所输出的信息进行标记。
  16. 一种血液分析系统，所述分析系统包括采样部、试剂供应部、反应部、检测部、控制器和处理器，其中：
    所述采样部，配置为从血液样本获取至少两份试样，并将所述试样分别输送到所述反应部；
    所述试剂供应部，配置为将所需的反应试剂供应到所述反应部；
    所述反应部，包括至少一个反应室，用于将第一试样与第一反应试剂混合制备第一混悬液，和用于将第二试样与第二反应试剂混合制备第二混悬液，且所述反应部配置为将第一混悬液和第二混悬液分别供应到所述检测部；
    所述检测部，包括电学检测部和光学检测部，其中所述电学检测部配置为使第一混悬液中粒子逐个通过电阻检测器，以获得第一混悬液中粒子的第一电信号，和所述光学检测部配置为使第二混悬液中的粒子逐个通过光学检测器，以获得第二混悬液中粒子的光学信息；
    所述控制器，所述控制器和所述采样部、试剂供应部、反应部和检测部偶联，控制所述采样部、试剂供应部、反应部和检测部的动作；和
    所述处理器，所述处理器和检测部耦合，其中
    所述处理器从所述检测部获取所述电信号和所述光学信息，根据所述电信号获得第一体积分布信息，根据所述光学信息获得白细胞分布信息，并利用前向散射光强度获得第二体积分布信息，和用所述第二体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
  17. 根据权利要求16所述的血液分析系统，其中所述电学检测部包括小孔阻抗检测器或者鞘流阻抗检测器。
  18. 根据权利要求16所述的血液分析系统，其中所述光学检测系统包括光源、允许细胞逐个通过的流动室、液路系统和散射光检测器。
  19. 根据权利要求18所述的血液分析系统，其中所述光学信息为散射光强度，优选地，所述光学信息包括前向散射光强度和侧向散射光强度。
  20. 根据权利要求18所述的血液分析系统，其中所述处理器根据所述散射光强度区分出白细胞粒子群，优选地，所述处理器进一步区分出嗜碱性白细胞亚群。
  21. 根据权利要求18所述的血液分析系统，其中所述光学检测部还包括荧光检测器，且所述第二反应试剂还含有荧光染料，或者所述试剂供应部配置为进一步将含有荧光染料的第三反应试剂供应到所述反应部的反应室中与所述第二试样进一步混合以制备第二混悬液。
  22. 根据权利要求21所述的血液分析系统，其中所述光学信息为至少一种散射光强度和荧光强度。
  23. 根据权利要求22所述的血液分析系统，其中所述散射光强度包括前向散射光强度，且所述处理器根据所述前向散射光强度和荧光强度区分出有核红细胞和白细胞粒子群，优选地，进一步区分出嗜碱性白细胞亚群。
  24. 根据权利要求22所述的血液分析系统，其中所述散射光强度包括侧向散射光强度，且所述处理器根据所述侧向散射光强度和荧光强度区分出淋巴细胞、单核细胞、中性粒细胞和嗜酸性粒细胞亚群。
  25. 一种血液分析系统，所述血液分析系统包括采样部、试剂供应部、反应部、检测部和控制器，其中：
    采样部，配置为用于从血液样本获取至少两份试样，并将所述试样分别输送到所述反应部；
    试剂供应部，配置为将所需的反应试剂供应到所述反应部；
    反应部，包括至少一个反应室，用于将第一试样与第一反应试剂混合制备第一混悬液，和用于将第二试样与第二反应试剂混合制备第二混悬液，且所述反应部配置为将第一混悬液和第二混悬液分别供应到所述检测部；
    检测部，包括电学检测部，其中所述电学检测部配置为使所述混悬液中粒子逐个通过电阻检测器，以获得第一混悬液的第一电信号，和第二混悬液的第二电信号；
    控制器，所述控制器和所述采样部、试剂供应部、反应部和检测部偶联，控制所述采样部、试剂供应部、反应部和检测部的动作；和
    处理器，所述处理器和检测部耦合，其中
    所述处理器从所述检测部获取所述第一和第二电信号，根据所述第一电信号获得第一体积分布信息，根据所述第二电信号获得第三体积分布信息，和用所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本的红细胞的校正体积分布信息。
  26. 根据权利要求16或25所述的血液分析系统，其中所述血液分析系统还包括输出装置。
  27. 根据权利要求26所述的血液分析系统，其中所述体积分布信息为直方图，优选地所述输出装置配置为输出所述校正的直方图，以及可选地输出第一和第二直方图或第一和第三直方图。
  28. 根据权利要求26所述的血液分析系统，其中所述处理器根据所述红细胞的校正体积分布信息获取选自如下组中的至少一个参数：红细胞计数、平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度变异系数(RDW-CV)、红细胞分布宽度标准差(RDW-SD)和红细胞压积(HCT)；和/或根据第二电信号获得白细胞总计数和白细胞的淋巴细胞、单核细胞和粒细胞亚群的计数；优选地，所述输出装置配置为输出所述参数。
  29. 根据权利要求27或28所述的血液分析系统，其中所述处理器将白细胞计数与第一阈值比较，当白细胞计数大于所述第一阈值时，或者所述处理器将校正前后的红细胞计数的差值与第二阈值比较，当所述差值大于所述第二阈值时，对所输出的红细胞相关信息进行标记。
  30. 一种计算机可读存储介质，所述计算机可读存储介质上存储有计算机程序，其特征在于，该计算机程序被处理器执行时实现以下步骤：
    获取通过检测第一混悬液获得的第一电信号，并根据所述第一电信号获得第一体积分布信息，其中所述第一混悬液由从血液样本获取的第一试样与第一反应试剂混合得到，且所述电信号通过使所述第一混悬液通过电学检测部的检测得到；
    获取通过检测第二混悬液获得的光学信息，并通过所述光学信息获得白细胞分布信息，并利用前向散射光强度获得第二体积分布信息，其中所述第二混悬液由从所述血液样本获取的第二试样与至少含有溶血剂的第二反应试剂混合得到，所述光学信息通过使所述第二混悬液通过光学检测部的检测得到，
    或者
    获取通过检测第二混悬液获得的第二电信号，并根据所述第二电信号获得第三体积分布信息，其中所述第二混悬液由从血液样本获取的第二试样与至少含有溶血剂的第二反应试剂混合得到，且所述电信号通过使所述第二混悬液通过所述电学检测部的检测得到；和
    用所述第二体积分布信息或所述第三体积分布信息校正所述第一体积分布信息，获得所述血液样本中红细胞的校正体积分布信息。
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CN113777009A (zh) * 2021-08-31 2021-12-10 深圳迈瑞动物医疗科技有限公司 适用于绵羊、山羊和奶牛的细胞分析仪及细胞检测方法
CN113777009B (zh) * 2021-08-31 2024-04-16 深圳迈瑞动物医疗科技股份有限公司 适用于绵羊、山羊和奶牛的细胞分析仪及细胞检测方法
WO2024001155A1 (zh) * 2022-06-29 2024-01-04 深圳安侣医学科技有限公司 基于显微放大数字图像的血液细胞分析方法及系统

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