WO2015039425A1 - 检测样本阴阳性的设备、方法及判断标准的确定方法 - Google Patents

检测样本阴阳性的设备、方法及判断标准的确定方法 Download PDF

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WO2015039425A1
WO2015039425A1 PCT/CN2014/074474 CN2014074474W WO2015039425A1 WO 2015039425 A1 WO2015039425 A1 WO 2015039425A1 CN 2014074474 W CN2014074474 W CN 2014074474W WO 2015039425 A1 WO2015039425 A1 WO 2015039425A1
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fluorescence intensity
sample
fluorescence
value
tested
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French (fr)
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胡鹏
雷霆
赵玉梅
谭伟峰
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Shenzhen Mindray Bio Medical Electronics Co Ltd
Beijing Shen Mindray Medical Electronics Technology Research Institute Co Ltd
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Shenzhen Mindray Bio Medical Electronics Co Ltd
Beijing Shen Mindray Medical Electronics Technology Research Institute Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
    • 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
    • G01N2015/1477—Multiparameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present application relates to a negative positive detection of antigen expression in an unknown sample, and more particularly to a method for determining a criterion for judging the positive expression of antigen in an unknown sample, a method for improving the accuracy of detection of a cell surface antigen expression, and a device for detecting a positive result of a sample.
  • HLA Human Leukocyte Antigen
  • MHC Major Histocompatibility Complex
  • the HLA-B27 antigen belongs to type I MHC and is expressed on all nucleated cells of the body, especially the surface of lymphocytes. In clinical studies, it is necessary to measure the expression of HLA-B27 antigen on the surface of leukocytes.
  • the HLA-B27 antigen belongs to the B7 cross-reaction group (C-HLA-B7, CREG HLA-B7) members, and the B7 cross-reactive family members include HLA-B27, HLA-B7, HLA-B42, HLA-B22, HLA-B40. And a series of antigens such as HLA-B 13.
  • the HLA-B27 monoclonal antibody corresponding to the HLA-B27 antigen can have different degrees of cross-reactivity with the B7 cross-reactive family members.
  • HLA-B27 antigen expression levels are mainly detected by: Complement dependent cytotoxicity (CDC), Enzyme-linked Immuno Sorbent Assay (ELISA), Polymerase chain reaction assay Polymerase Chain Reaction (PGR) and Flow Cellometry (FCM).
  • CDC Complement dependent cytotoxicity
  • ELISA Enzyme-linked Immuno Sorbent Assay
  • PGR Polymerase chain reaction assay Polymerase Chain Reaction
  • FCM Flow Cellometry
  • FCM is mainly a monoclonal antibody corresponding to the fluorescently labeled HLA-B27 antigen, and lymphocytes
  • the surface of the HLA-B27 antigen binds to the lymphocytes to emit a certain fluorescence.
  • the fluorescence intensity value of this fluorescence can be measured by flow cytometry.
  • the presence or absence of HLA-B27 antigen on the lymphocyte surface is determined by the measured fluorescence intensity. , thereby judging the positive of the sample.
  • a method for detecting HLA-B27 antigen expression by FCM is: using FITC-HLA-B27/PE-CD3 two-color reagent and sample reaction, analyzing the fluorescence value of FITC channel of CD3 positive cells by flow cytometry, and fluorescence The median value of the value is compared with the preset threshold value on the reagent bottle to determine the positive yin of the sample.
  • the method is simpler to operate, but the result is sometimes not accurate enough. Summary of the invention
  • a method for determining a criterion for judging an abnormality of an unknown sample includes:
  • the fluorescent detection reagent includes An antibody labeled with a fluorescent substance corresponding to the antigen of interest
  • the fluorescence signal of the first fluorescent substance emitted by each cell in each test sample liquid is detected by a flow cytometer to obtain a first fluorescence intensity value
  • an optimum value is determined from the difference between the plurality of test samples as a fluorescence threshold for judging the negative positive of the unknown sample.
  • a method for improving the accuracy of cell surface antigen expression detection comprising:
  • the sample to be tested is mixed with the fluorescent detection reagent to obtain a sample liquid to be tested, and the fluorescent detection reagent includes an antibody corresponding to the antigen of interest labeled with the first fluorescent substance;
  • the sample liquid is detected by a flow cytometer, and the fluorescence signal of the first fluorescent substance emitted by each cell in the sample liquid is detected to obtain a first fluorescence intensity value; Calculating the fluorescence intensity of the sample liquid according to the first fluorescence intensity value of the cell of interest in the sample liquid to be tested; and
  • the difference between the fluorescence intensity of the test sample liquid and the fluorescence intensity of the isotype control is calculated, and the difference is used to compare with a preset fluorescence threshold.
  • An apparatus for detecting expression of a cell surface antigen comprising:
  • a sample preparation system for mixing the sample of the body fluid to be tested with the fluorescence detection reagent to obtain a sample liquid to be tested, wherein the fluorescence detection reagent comprises an antibody corresponding to the antigen of interest labeled with the first fluorescent substance;
  • a flow cytometer for detecting a sample liquid to be tested, wherein the flow cytometer detects a fluorescence signal of a first fluorescent substance emitted by each cell in the sample liquid to be measured, to obtain a first fluorescence intensity value;
  • a data processing system configured to receive data output by the flow cytometer, calculate a fluorescence intensity of the sample liquid according to a first fluorescence intensity value of the cell of interest in the sample liquid to be tested, and calculate a fluorescence intensity of the sample liquid to be tested and an isotype control The difference in fluorescence intensity, which is used to compare with a preset fluorescence threshold.
  • the cell surface antigen fluorescence threshold is increased, the interference of the non-specific binding on the detection result is reduced, and the accuracy of the HLA-B27 antigen expression detection result is improved.
  • FIG. 1 is a schematic view showing the structure of a cell surface antigen expression detecting apparatus according to an embodiment
  • FIG. 2 is a flow chart of determining a determination criterion of an embodiment
  • Figure 3 is a flow chart showing the detection of cell surface antigen expression in an embodiment
  • Figure 4 is a scatter diagram of the scattered light of the sample 1 to be tested
  • Figure 5 is a isotype control scatter plot of the sample 1 to be tested;
  • Figure 6 is a scatter plot of the sample to be tested for sample 1
  • Figure 7 is a scatter plot of the scattered light of the sample 2 under test.
  • Figure 8 is a homograph scatter plot of the sample 2 being tested.
  • Figure 9 is a scatter plot of the sample to be tested for sample 2 to be tested.
  • Cell surface antigens are usually distributed on the surface of nucleated cells in various tissues. It is found by analysis that fluorescently labeled monoclonal antibodies bind non-specifically to HLA-B27 antigen on the surface of lymphocytes, and this non-specific binding to HLA-B27 antigen Positive or negative judgments can cause interference. For example, some lymphocyte surface HLA-B27 antigens are truly negative, and due to the above non-specific binding, these lymphocyte 7 surface HLA-B27 antigens are detected positive, thereby reducing the white blood cell surface. The accuracy of antigen detection. In the examples of the present application, the expression of cell surface antigen was determined by flow cytometry.
  • the schematic diagram of the apparatus for detecting cell surface antigen expression by flow cytometry is shown in FIG. 1, and may include a sample preparation system 101, a flow cytometer, and a data processing system 104, a sample.
  • the liquid preparation system 101 is configured to mix the sample of the body fluid to be tested with the fluorescence detection reagent to obtain the sample liquid to be tested after the dyeing.
  • the flow cytometer is used for detecting the sample liquid to be tested.
  • the flow cytometer usually includes a light detecting unit 102 and a flow chamber 103.
  • the sample liquid to be tested passes through the detection area of the flow chamber 103 in a single cell manner under the sheath liquid sheathing.
  • the detecting unit 102 is for collecting light emitted by each cell particle.
  • the light detecting unit 102 includes at least one detecting channel including a fluorescence detecting channel for detecting fluorescence emitted by the cell after being labeled by the fluorescent substance.
  • the fluorescence detecting channel may include a plurality of fluorescent detecting channels, such as first and second channels, the filter band of each channel and the fluorescence detected thereby The light emission band of the substance is the same.
  • the sample liquid prepared by the sample preparation system 101 can be delivered to the flow chamber 103 of the flow cytometer through the delivery system 105.
  • the data processing system 104 is configured to receive data output by the flow cytometer and process the data.
  • the sample of the body fluid to be tested may be blood or other body fluid such as urine. At the time of detection, an antigen is usually used as an antigen of interest, and the sample to be tested is judged to be positive for the antigen of interest.
  • the HLA-B27 antigen is used as an antigen of interest, and a blood sample is taken as an example.
  • a method for determining a criterion for judging an unknown sample is positive, and the criterion includes a fluorescence threshold value and a ratio threshold value.
  • FIG. 2 is a flow chart for determining the judgment standard. The method includes the following steps:
  • step 10 a plurality of test samples are selected, which are known to be positive for the HLA-B27 antigen. Each test sample was processed according to steps 11-13. For ease of description, only one test sample is described in steps 11-13.
  • Step 11 Mixing the test sample with the fluorescent detection reagent by using the sample liquid preparation system to prepare the required test sample liquid.
  • the fluorescent detection reagent includes an antibody against the HLA-B27 antigen labeled with the first fluorescent substance (hereinafter referred to as HLA-B27 antibody) and an antibody corresponding to the interference antigen of the antigen of interest labeled with the second fluorescent substance (hereinafter referred to as an interfering antibody).
  • the HLA-B27 antibody may be, for example, a monoclonal antibody
  • the second fluorescent substance and the first fluorescent substance are fluorescent substances such as fluorescein or fluorescent protein having different emission wavelengths.
  • the first fluorescent substance is FITC
  • the second fluorescent substance is PE.
  • the first fluorescent substance is PE and the second fluorescent substance is FITC.
  • the interfering antigen is the other antigen of its family, mainly HLA-B7.
  • the fluorescently labeled HLA-B27 antibody binds to the HLA-B27 antigen, and is bound by the fluorescently labeled interfering antibody and the interfering antigen (for example, HLA-B7 antigen), thus carrying HLA-B27.
  • the antigen and leukocytes that interfere with the antigen are stained with a fluorescent substance, and the fluorescently labeled HLA-B27 antibody also cross-reacts with an interfering antigen (for example, HLA-B7 antigen).
  • an interfering antigen for example, HLA-B7 antigen
  • the test sample solution is detected by flow cytometry.
  • the fluorescence emitted by the cells by the fluorescent substance is detected by a specific fluorescence detecting channel.
  • the light detecting unit of the flow cytometer may include a plurality of light detecting channels according to the needs of the rear end light collection, and a plurality of The photodetectors of the light detecting channels may be independent or integrated.
  • the first channel is configured to detect fluorescence emitted by the first fluorescent substance, and the wavelength detectable by the photodetector of the channel includes an emission wavelength of the first fluorescent substance, so that the fluorescence emitted by the cell labeled by the first fluorescent substance can be
  • a channel is collected to obtain a first fluorescence intensity value.
  • the second channel is configured to detect fluorescence emitted by the second fluorescent substance, and the wavelength detectable by the photodetector of the channel includes an emission wavelength of the second fluorescent substance, so that the fluorescence emitted by the cell labeled by the second fluorescent substance can be second. Channel collection, obtaining a second fluorescence intensity value.
  • Each cell labeled with a fluorescent substance corresponds to a first fluorescence intensity value and a second fluorescence intensity value.
  • the scattered light emitted by the cells by the light beam may be detected in this step, and therefore, the detection channel is further Includes scattered light detection channels.
  • the detected scattered light includes forward scattered light that reflects the size of the cell volume and side scattered light that reflects the internal complexity of the cell.
  • the flow cytometer converts the detected various optical signals into electrical signals for transmission to the data processing system.
  • Step 13 The data processing system processes various data detected by the flow cytometer, and calculates a difference value and a ratio of each sample liquid, wherein the ratio is a ratio of the number of cells falling into the control region to the total number of cells, and the ratio may be, for example, A percentage, or a decimal equal to the percentage.
  • the calculation steps of the difference include:
  • Step 131 Calculate a fluorescence intensity of the test sample liquid according to the first fluorescence intensity value.
  • the fluorescence intensity of the sample solution it can be calculated based on the first fluorescence intensity value of the cells of interest in the test sample solution, and the cells of interest may be all white blood cells or only lymphocytes.
  • the fluorescence intensity of the test sample solution can be calculated based only on the first fluorescence intensity value of the lymphocytes, and the specific steps include: 12 Collected scattered light, classify and count white blood cells, identify lymphocytes, obtain the first fluorescence intensity value of lymphocytes, and calculate the fluorescence intensity of the test sample liquid according to the first fluorescence intensity values of these lymphocytes.
  • the fluorescence intensity may be an average of these first fluorescence intensity values or a median value of these first fluorescence intensity values.
  • step 132 the difference of the test sample liquid is calculated.
  • the fluorescence intensity of the test sample solution is obtained from the isotype control data, and the isotype control data includes the fluorescence intensity of the isotype control.
  • the fluorescence intensity of the isotype control may be a pre-stored value in the device, or may be the fluorescence intensity of the isotype control sample solution.
  • split the test sample into two One part is used to prepare the test sample liquid, and the other one is used to prepare the isotype control sample liquid, that is, the isotype control reagent of the fluorescent detection reagent is mixed with another test sample to obtain the isotype control sample liquid of the test sample liquid,
  • the first fluorescent intensity value of each cell of the isotype control sample solution is obtained by using a flow cytometer to detect the isotype control sample solution, and the fluorescence intensity of the isotype control is calculated according to the first fluorescence intensity value of the cell of interest in the isotype control sample solution.
  • the difference between the fluorescence intensity of the test sample solution and the fluorescence intensity of the isotype control was calculated as the difference between the test sample liquids.
  • the calculation steps of the ratio include:
  • Step 133 generating a homotype control scatter plot.
  • Obtaining isotype control data, wherein the isotype control data includes a first fluorescence intensity value and a second fluorescence intensity value of the cell of interest in the isotype control sample solution, according to the first fluorescence intensity value and the second fluorescence intensity of the cell of interest in the isotype control sample solution The values form a two-dimensional isotype control scatter plot.
  • the scattergram is a plurality of data sets of a two-dimensional array formed by the first fluorescence intensity value and the second fluorescence intensity value, or a two-dimensional map formed according to the first fluorescence intensity value and the second fluorescence intensity value, for example,
  • the first fluorescence intensity value is the abscissa and the second fluorescence intensity value is the ordinate, and vice versa.
  • Each point in the figure represents a thin moon packet.
  • Step 134 setting a gate in the isotype control scatter plot.
  • Setting a door means setting conditions to classify cells that meet certain conditions into one class.
  • a predetermined proportion of cells are distributed in a designated region of the threshold.
  • a first threshold and a second threshold are set, and the designated region is a region where the first fluorescence intensity value is less than the first threshold and the second fluorescence intensity value is less than the second threshold, then the first threshold and a second threshold Forming a threshold that causes the ratio of the total number of cells in the cell occupying dot map falling within the designated region to satisfy a predetermined ratio, that is, the threshold is such that the ratio of the total number of cells in the cell occupying the specified region is Is a predetermined ratio, or within an acceptable error range of the predetermined ratio, the predetermined ratio may be a value preset according to actual requirements, for example, the ratio of the total number of cells in the cell occupying the designated area. It is 95% or 98% or 98% ⁇ 0.5%, etc.
  • Step 135 generating a scatter plot of the test sample.
  • a first fluorescence intensity value and a second fluorescence intensity value of the cells of interest in the test sample solution are obtained, and a test sample scattergram is formed according to the first fluorescence intensity value and the second fluorescence intensity value of the cells of interest in the test sample solution.
  • step 137 the cell population is divided by the same threshold in the scatter plot of the test sample, and the calculation falls into the designation.
  • the number of cells in the control region of the region is the ratio of the total number of cells in the scatter plot of the test sample.
  • the control area is an area different from the designated area in the positive direction along the first fluorescence intensity value, for example, the designated area is the area where the first fluorescence intensity value is smaller than the first threshold and the second fluorescence intensity value is smaller than the second threshold, then the comparison area is A region where the fluorescence intensity value is greater than the first threshold and the second fluorescence intensity value is less than the second threshold.
  • the threshold is set to the cross door
  • the designated area is the lower left quadrant of the cross door
  • the control area is the lower right quadrant of the cross door.
  • step 14 the positive samples of all the test samples and the difference and ratio of each sample are counted, and one of the plurality of test samples is determined according to the known negative-positive and acceptable sensitivity and specificity criteria of the test sample.
  • the difference and ratio of the sample are used as the threshold value for the fluorescence and the ratio threshold for judging the positive sample of the unknown sample.
  • acceptable sensitivity and specificity criteria are determined according to user requirements and specific equipment.
  • the flow cytometer prior to detecting the test sample solution by flow cytometry, is calibrated to adjust the fluorescence value of the flow cytometer, and the calibration fluorescent microsphere is displayed on the fluorescence detection channel. The fluorescence value is adjusted to the calibration value.
  • the following is an example to further illustrate how to determine the HLA-B27 negative-positive judgment criteria for unknown samples.
  • the HLA-B27 detection reagent i.e., a fluorescent detection reagent for detecting HLA-B27 antigen
  • the HLA-B27 detection reagent is mixed with a blood sample, and reacted at room temperature for a while to stain the sample.
  • the stained sample is detected by flow cytometry.
  • the flow cytometer is calibrated before the sample is detected, and the channel voltage of the first channel FL1 and the second channel FL2 of the flow cytometer is adjusted, and the calibration fluorescence is adjusted according to the parameters of the first fluorescent substance FITC and the second fluorescent substance PE.
  • the fluorescence value displayed by the microspheres on the FITC/PE channel is adjusted to the calibration value.
  • the lymphocytes in the sample can be identified by the forward scattered light signal and the side scattered light signal, and the fluorescence value signals of the FL1 and FL2 channels of the lymphocytes are collected.
  • MFI (difference) MFI (FITC- HLA- B27) - MFI (FITC- IgG2a)
  • MFI FITC-HLA-B27
  • MFI FITC-IgG2a
  • MFI-IgG2 is the fluorescence intensity of the isotype control
  • MFI-B27 is the fluorescence intensity of the sample
  • MFI (difference) is the difference between MFI-B27 and MFI-IgG2.
  • Q4 is the percentage of lymphocytes in the lower right quadrant of the cross-gate of the FITC-HLA-B27/PE- HLA-B7 scatter plot.
  • Step 21 mixing the blood sample to be tested and the fluorescent detection reagent by using a sample liquid preparation system to prepare a required sample liquid to be tested.
  • the fluorescent detection reagent includes an HLA-B27 antibody labeled with a first fluorescent substance and an interference antibody labeled with a second fluorescent substance.
  • the HLA-B27 antibody may be, for example, a monoclonal antibody, and the second fluorescent substance and the first fluorescent substance are fluorescent substances having different emission wavelengths.
  • the fluorescently labeled HLA-B27 antibody and the HLA-B27 antigen are combined, and the fluorescently labeled interference antibody and the perturbation antigen (for example, HLA-B7 antigen) are combined, thereby carrying
  • the white blood cells of the HLA-B27 antigen and the perturbation antigen are stained with a fluorescent substance, and the fluorescently labeled HLA-B27 antibody also cross-reacts with an interfering antigen (for example, HLA-B7 antigen).
  • the label that has not been bound to the leukocytes is removed by washing and separation to obtain a sample liquid to be tested after staining.
  • step 22 the sample liquid to be tested is detected by flow cytometry.
  • the specific detection step can be the same as the detection step in the first embodiment.
  • the first fluorescence intensity value and the second fluorescence intensity value of the labeled cells in the sample liquid to be tested are detected, and the scattered light emitted by the cells by the light beam can be detected as needed.
  • the flow cytometer converts the detected various optical signals into electrical signals for transmission to the data processing system.
  • Step 23 The data processing system processes various data detected by the flow cytometer, and calculates a difference and a ratio of the sample liquids to be tested.
  • the calculation steps of the difference include:
  • Step 231 Calculate a fluorescence intensity of the sample liquid to be tested according to the first fluorescence intensity value.
  • the cells of interest may be all white blood cells or only lymphocytes.
  • the step of identifying the lymphocytes includes: using the scattered light collected in step 22, classifying and counting the white blood cells, identifying the lymphocytes, obtaining the first fluorescence intensity value of the lymphocytes, and calculating the measured value according to the first fluorescence intensity values of the lymphocytes.
  • the fluorescence intensity of the sample solution In a preferred embodiment, the cells of interest in this step should be determined with the criteria for judgment.
  • the fluorescence intensity may be an average of these first fluorescence intensity values or a median value of these first fluorescence intensity values.
  • the fluorescence intensity in this step should also be consistent with the algorithm for determining the fluorescence intensity during the standard determination process.
  • Step 232 Calculate the difference of the sample liquid to be tested.
  • the fluorescence intensity of the sample liquid to be tested is obtained from the isotype control data.
  • the isotype control data includes the fluorescence intensity of the isotype control.
  • the fluorescence intensity of the isotype control may be a pre-stored value in the device or the fluorescence intensity of the isotype control sample solution.
  • the test sample is divided into two parts, one for preparing the sample liquid to be tested, and the other for preparing the isotype control sample liquid, that is, the same type of control reagent of the fluorescence detection reagent and another sample to be tested are mixed and processed.
  • the isotype control sample solution of the sample liquid to be tested is detected by flow cytometry using the same method as the sample liquid to be tested, and the fluorescence intensity of the isotype control sample liquid is obtained.
  • the difference between the fluorescence intensity of the sample liquid to be tested and the fluorescence intensity of the isotype control is taken as the difference of the sample liquid to be tested.
  • the calculation steps of the ratio include:
  • Step 233 generating a homotype control scatter plot.
  • Step 234 setting a threshold in the isotype control scatter plot, the threshold is such that the ratio of the total number of cells in the cell occupying point map falling within the specified area should satisfy a predetermined ratio, that is, the threshold causes the cells falling into the designated area to occupy
  • the ratio of the total number of cells in the scattergram should be a predetermined ratio, or within an acceptable error range of the predetermined ratio, and the predetermined ratio may be a value preset according to actual requirements, for example, cells falling into the designated area.
  • the ratio of the total number of cells in the scatter plot is equal to 95%, 98% or a value in the vicinity.
  • Judgment step 235 generating a scatter plot of the sample to be tested.
  • step 237 the cell population is divided by the same threshold in the scatter plot of the sample to be tested, and the ratio of the number of cells in the control region falling into the designated region to the total number of cells in the scatter plot of the sample to be tested is calculated.
  • the threshold is set to the cross door
  • the designated area is the lower left quadrant of the cross door
  • the control area is the lower right quadrant of the cross door.
  • the difference and the ratio are compared with the fluorescence threshold and the ratio threshold, respectively, and the sample to be tested is positive for the HLA-B27 antigen based on the comparison result.
  • the judgment may be that the operator compares the difference value and the ratio of the output of the data processing system with the fluorescence threshold value and the ratio threshold value, or may input the fluorescence threshold value and the ratio threshold value into the data processing system in advance, and the data processing system is After calculating the difference and ratio of the sample to be tested, the difference and the ratio are automatically compared with the fluorescence threshold and the ratio threshold.
  • the judgment rule may be, for example,: when the difference of the sample to be tested is greater than or equal to the fluorescence threshold, and the ratio of the sample to be tested is greater than or equal to the threshold value, the sample liquid to be tested is positive, otherwise it is negative.
  • any one of the judgment rules "greater than or equal to” can also be changed to "greater than”.
  • the fluorescence intensity of the isotype control is used as the base, so It reduces the interference of non-specific binding on the detection results and improves the accuracy of the detection results of HLA-B27 antigen expression.
  • the fluorescence intensity of B27 is greater than the critical value, and the fluorescence intensity of B7 is less than the critical value, and the fluorescence intensity greater than the critical value should be the cell that actually expresses the B27 antigen. Therefore, according to the result of Q4 of the unknown sample tested, the expression of HLA-B27 antigen can be accurately determined, thereby eliminating the influence of HLA-B7 antigen on the detection result, and significantly reducing the false positive of the detection result.
  • the fluorescence calibration of the flow cytometer can achieve good mutual migration between different machines and different flow models, and the test results are very comparable between different laboratories.
  • the known HLA-B27-negative fresh anticoagulant was used as the test sample 1 and HLA-B27 to detect the two-color reagent.
  • the prepared sample was detected by flow cytometry, using forward scattered light (FSC) and side scatter.
  • Light (SSC) selects lymphocytes. As shown in Fig. 4, in the scattergram formed by forward scattered light (FSC) and side scattered light (SSC), lymphocytes are in the P1 region in the lower left corner.
  • the FL1/FL2 scatter plot cross gate such as As shown in Figure 5, assume the bottom left of the cross door The quadrant is the designated area and requires 98% of the cells to fall into the lower left quadrant.
  • FIG. 6 shows a scatter plot of the fluorescence intensity values of the first FL 1 and the second channel FL2 of the sample 1 to be tested. In the scatter plot of the sample 1 to be tested, the same as in the isotype scatter plot.
  • the cross gate calculate the MFI (difference) value and Q4 value of the sample 1 to be tested, the Q4 value is the percentage of the number of cells falling into the fourth quadrant of the cross gate, and the fourth quadrant is the third. Quadrant control quadrant.
  • the known HLA-B27-positive fresh anticoagulant was used as the test sample 2 and HLA-B27 to detect the two-color reagent reaction, and the lymphocytes were selected by forward scattered light (FSC) and side scattered light (SSC), as shown in the figure. As shown in Fig. 7, in the scattergram formed by forward scattered light (FSC) and side scattered light (SSC), lymphocytes are in the lower left corner P1 region.
  • the number of cells distributed to the left of the first threshold boundary line and the number of cells distributed under the second threshold boundary line are still 99%, and the first threshold boundary line and the second threshold boundary line form a "word gate, the cross
  • the number of cells distributed in the lower left quadrant of the gate accounts for 98% of the total number of cells in the scatter plot. As shown in Fig.
  • the scatter plot of the fluorescence intensity values of the first FL1 and the second FL2 of the sample 2 to be tested is In the scatter plot of sample 2, the same cross gate as in the isotype scatter plot is used to calculate the MFI (difference) value and the Q4 value of the sample 2 to be tested, and the detection result is MFI (difference) value > 540 and Q4 > 80%, the test result is positive.
  • the criterion includes a fluorescence threshold value and a ratio threshold value. In other embodiments, the criterion may include only one of a fluorescence threshold value and a ratio threshold value, and correspondingly, the unknown sample is performed. In the detection of HLA-B27 antigen expression, it is only necessary to compare with a criterion.
  • the antigen of interest is an HLA-B27 antigen and the interfering antigen is an HLA-B7 antigen.
  • the antigen of interest may also be other cell surface antigens, and the corresponding interfering antigen is The cell surface antigen has a cross-reactive antigen.
  • the antibody used is a monoclonal antibody, and in other embodiments, a polyclonal antibody may also be used.

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Abstract

一种用于判断未知样本阴阳性的判断标准的确定方法、提高细胞表面抗原表达检测准确性的方法以及检测样本阴阳性的设备，将被测样本液与荧光检测试剂混合处理，得到染色后的被测样本液，采用流式细胞仪检测被测样本液，得到被测样本液的荧光强度，计算被测样本液的荧光强度与同型对照的荧光强度的差值，将差值与预设的荧光临界值进行比较，根据比较结果判断细胞表面抗原的表达。通过确定适合的荧光临界值减少了非特异性结合对检测结果的干扰，提高了检测HLA-B27抗原表达的准确性。

Description

说明书
检测样本阴阳性的设备、 方法及判断标准的确定方法 技术领域
本申请涉及未知样本抗原表达阴阳性检测, 尤其涉及一种用于判断未知样本 中抗原表达阴阳性的判断标准的确定方法、提高细胞表面抗原表达检测准确性的 方法以及检测样本阴阳性的设备。 背景技术
人类白细胞表面抗原 I Human Leukocyte Antigen, HLA )是人类主要组织相 容性复合体 ( Major Histocompatibility Complex, MHC ) 的表达产物， 在免疫系 统中主要负责细胞之间的相互识别和诱导免疫反应， 调节免疫应答的功能。 根据 HLA结构、 功能和组织分布的不同可分为三类： I类分子为 HLA-A、 HLA-B及 HLA-C 系列抗原， 其广泛分布于各组织有核细胞表面； II类分子为 HLA-DR、 HLA- DP及 HLA-DQ系列抗原， 其主要在 B淋巴细胞和抗原呈递细胞上表达； ΠΙ类分子为补体成分。
HLA-B27抗原属于 I型 MHC, 基本上表达在机体所有有核细胞上, 尤其是 淋巴细胞表面含量丰富, 在临床研究上需要测量白细胞表面 HLA-B27抗原的表 达。 HLA-B27抗原属于 B7交叉反应族 ( Cross-reaction Group HLA-B7, CREG HLA-B7 )成员，B7交叉反应族成员包括 HLA- B27、HLA-B7、HLA-B42、HLA- B22、 HLA-B40以及 HLA-B 13等一系列抗原。 HLA-B27抗原对应的 HLA-B27单克隆 抗体均可与 B7交叉反应族成员有不同程度的交叉反应。
目前， HLA-B27 抗原表达水平检测的方法主要有： 补体依赖的细胞毒试验 ( Complement dependent cytotoxicity, CDC )、 酶联免疫测定试验 ( Enzyme-linked Immuno Sorbent Assay , ELISA )、 聚合酶链式反应检测法 ( Polymerase Chain Reaction, PGR ) 以及流式细胞法 I Flow Cytometry, FCM )。 FCM因其无需分离 淋巴细胞， 操作简单且具有很高的灵敏度和特异性， 因此被广泛使用。
FCM主要是利用荧光标记的 HLA-B27抗原对应的单克隆抗体， 与淋巴细胞 表面的 HLA-B27抗原结合, 使淋巴细胞发出一定的荧光， 这种荧光的荧光强度 值可被流式细胞仪测定, 通过所测定的荧光强度值的高低判断淋巴细胞表面是否 存在 HLA-B27抗原 , 从而判断样本的阴阳性。 一种釆用 FCM检测 HLA-B27抗 原表达的方案是： 采用 FITC-HLA- B27/PE-CD3双色试剂和样本反应， 通过流式 细胞仪分析 CD3阳性细胞 FITC通道荧光值的强弱，将荧光值的中位值和试剂瓶 上预设的临界值比较来判定样本的阴阳性， 该方法操作较简单， 但结果有时不够 准确。 发明内容
基于此， 有必要提供一种能提高细胞表面抗原表达检测结果的准确性的用于 判断未知样本阴阳性的判断标准的确定方法、提高细胞表面抗原表达检测准确性 的方法以及检测样本阴阳性的设备。
一种用于判断未知样本阴阳性的判断标准的确定方法， 包括：
将多个试验样本分别和荧光检测试剂混合处理， 得到各自对应的试验样本 液， 所述多个试验样本针对于感兴趣抗原已知被确定为阴性或阳性， 所述荧光检 测试剂中包括采用第一荧光物质标记的与感兴趣抗原对应的抗体；
采用流式细胞仪检测各试验样本液中各细胞发出的第一荧光物质的荧光信 号， 获得第一荧光强度值；
根据所述试验样本液中感兴趣细胞的第一荧光强度值， 计算该试验样本液的 荧光强度；
计算所述试验样本液的荧光强度与同型对照的荧光强度的差值；
根据试验样本的已知的阴阳性， 从多个试验样本的差值中确定出一个最合适 值作为判断未知样本阴阳性的荧光临界值。
一种提高细胞表面抗原表达检测准确性的方法， 包括：
将被测样本与荧光检测试剂混合处理， 得到染色后的被测样本液， 荧光检测 试剂中包括采用第一荧光物质标记的与感兴趣抗原对应的抗体；
采用流式细胞仪检测被测样本液，检测被测样本液中各细胞发出的第一荧光 物质的荧光信号， 获得第一荧光强度值； 根据被测样本液中感兴趣细胞的第一荧光强度值计算被测样本液的荧光强 度; 及
计算被测样本液的荧光强度与同型对照的荧光强度的差值， 所述差值用于与 预设的荧光临界值进行比较。
一种用于检测细胞表面抗原表达的设备， 包括：
样本液制备系统， 用于将被测体液样本与荧光检测试剂混合处理， 得到染色 后的被测样本液， 荧光检测试剂中包括釆用第一荧光物质标记的与感兴趣抗原对 应的抗体；
流式细胞仪， 用于检测被测样本液， 所述流式细胞仪检测被测样本液中各细 胞发出的第一荧光物质的荧光信号， 获得第一荧光强度值； 及
数据处理系统， 用于接收流式细胞仪输出的数据， 根据被测样本液中感兴趣 细胞的第一荧光强度值计算被测样本液的荧光强度， 计算被测样本液的荧光强度 与同型对照的荧光强度的差值， 所述差值用于与预设的荧光临界值进行比较。
在上述用于判断未知样本阴阳性的判断标准的确定方法、提高细胞表面抗原 荧光临界值， 减少了非特异性结合对检测结果的干扰， 提高了对 HLA- B27抗原 表达检测结果的准确性。
为了更清楚地说明本发明实施例， 下面将对实施例中所需要使用的附图作简 单地介绍， 显而易见地， 下面描述中的附图仅仅是本发明的一些实施例， 对于本 领域普通技术人员来讲， 在不付出创造性劳动的前提下， 还可以根据这些附图获 得其他的附图。
图 1为一实施方式的检测细胞表面抗原表达设备的结构示意图；
图 2为一实施方式的确定判断标准的流程图；
图 3为一实施方式的检测细胞表面抗原表达的流程图；
图 4为被测样本 1的散射光的散点图；
图 5为被测样本 1的同型对照散点图； 图 6为被测样本 1的被测样本散点图
图 7为被测样本 2的散射光的散点图
图 8为被测样本 2的同型对照散点图； 及
图 9为被测样本 2的被测样本散点图。 具体实施方式
以下说明提供了用于完全理解各个实施例以及用于本领域的技术人员实施 的特定细节。 然而， 本领域的技术人员应该理解， 无需这样的细节亦可实践本发 明。 在一些实例中， 为了避免不必要地混淆对实施例的描述， 没有详细示出或描 述公知的结构和功能。除非上下文清楚地要求， 否则，贯穿本说明书和权利要求， 用语"包括"、 "包含"等应以包含性的意义来解释而不是排他性或穷尽性的意义， 即， 其含义为"包括， 但不限于"。 在本详细描述部分中， 使用单数或复数的用语 也分别包括复数或单数。
细胞表面抗原通常分布于各组织中的有核细胞表面 , 通过分析发现， 荧光标 记的单克隆抗体与淋巴细胞表面 HLA-B27抗原存在非特异性结合， 而这种非特 异性结合对 HLA-B27抗原的阳性或阴性的判断会造成干扰, 例如， 某些淋巴细 胞表面 HLA-B27抗原真实呈阴性， 而由于上述非特异性结合， 使得这些淋巴 7 细胞表面 HLA-B27抗原检测为阳性，从而降低了白细胞表面抗原检测的准确度。 本申请实施例中， 采用流式细胞法判断细胞表面抗原的表达。 在一实施方式中， 这种釆用流式细胞法来检测细胞表面抗原表达的设备的结构示意图如图 1所示， 可包括样本液制备系统 101、 流式细胞仪和数据处理系统 104， 样本液制备系统 101用于将被测体液样本与荧光检测试剂混合处理， 得到染色后的被测样本液。 流式细胞仪用于检测被测样本液， 流式细胞仪通常包括光检测单元 102和流动室 103 , 被测样本液在鞘液的裹挟下以单细胞方式通过流动室 103 的检测区域， 光 检测单元 102用于收集各细胞粒子发出的光。 光检测单元 102包括至少一个检测 通道， 其中包括荧光检测通道， 荧光检测通道用于检测因细胞被荧光物质标记后 发出的荧光。 为了将不同荧光物质发出的荧光区别开来， 荧光检测通道可以包括 多个荧光检测通道， 例如第一和第二通道， 每个通道的滤光波段与其检测的荧光 物质的发光波段相同。样本液制备系统 101制得的样本液可通过输送系统 105输 送到流式细胞仪的流动室 103。 数据处理系统 104用于接收流式细胞仪输出的数 据， 对数据进行处理。 被测体液样本可以是血液， 也可以是尿液等其它体液。 在 检测时通常以某一抗原为感兴趣抗原 , 判断被测样本针对于该感兴趣抗原的阴阳 性。
下面以 HLA- B27抗原为感兴趣抗原、 以血液样本为例进行说明。
在一实施方式， 用于判断未知样本阴阳性的判断标准的确定方法， 判断标准 包括荧光临界值和比值临界值。 请参考图 2, 其为确定判断标准的流程图。 该方 法包括以下步骤：
步骤 10, 选择多个试验样本, 该多个试验样本针对于 HLA-B27抗原已知其 阴阳性。 将每个试验样本按照步骤 11-13进行处理。 为方便描述， 在步骤 11-13 中只针对一个试验样本进行描述。
步骤 11 , 采用样本液制备系统将试验样本分别和荧光检测试剂混合处理， 制 成需要的试验样本液。 荧光检测试剂中包括采用第一荧光物质标记的 HLA- B27 抗原的抗体（以下简称 HLA- B27抗体）和采用第二荧光物质标记的与感兴趣抗 原的干扰抗原对应的抗体（以下简称干扰抗体）。 HLA-B27抗体例如可以是单克 隆抗体， 第二荧光物质与第一荧光物质为不同发光波长的荧光素或荧光蛋白等荧 光物质， 例如， 第一荧光物质为 FITC, 第二荧光物质为 PE, 或者第一荧光物质 为 PE, 第二荧光物质为 FITC。 对于 HLA-B27抗原来说, 干扰抗原是与它同族 的其它抗原， 主要是 HLA-B7。 当将荧光检测试剂和试验样本混合后， 被荧光标 记的 HLA-B27抗体和 HLA-B27抗原结合，被荧光标记的干扰抗体和干扰抗原（例 如 HLA-B7抗原 )结合， 因此携带有 HLA-B27抗原和干扰抗原的白细胞被荧光 物质染色， 同时被荧光标记的 HLA-B27抗体也与干扰抗原 (例如 HLA-B7抗原 ) 发生交叉反应。 通过清洗分离， 将未与白细胞结合的标记物清除， 得到染色后的 试验样本液„
步骤 12,采用流式细胞仪检测试验样本液。 当试验样本液通过流式细胞仪的 流动室时， 通过特定的荧光检测通道检测细胞因被荧光物质标记而发出的荧光。 流式细胞仪的光检测单元根据后端光收集的需要可以包括多个光检测通道， 多个 光检测通道的光检测器可以是各自独立， 也可以是合一的。 其中第一通道用于检 测第一荧光物质发出的荧光， 该通道的光检测器可检测的波长包括第一荧光物质 的发光波长， 以使被第一荧光物质标记的细胞发出的荧光可被第一通道收集 , 获 得第一荧光强度值。 第二通道用于检测第二荧光物质发出的荧光， 该通道的光检 测器可检测的波长包括第二荧光物质的发光波长 , 以使被第二荧光物质标记的细 胞发出的荧光可被第二通道收集， 获得第二荧光强度值。 每个被荧光物质标记的 细胞对应一个第一荧光强度值和一个第二荧光强度值。 在本步骤中， 除了检测细 胞因被荧光物质标记而发出的荧光外， 为了减少后续的数据处理量， 还可以在本 步骤中检测细胞因被光束照射而发出的散射光， 因此， 检测通道还包括散射光检 测通道。 在一种具体实例中， 检测的散射光包括反映细胞体积大小的前向散射光 和反映细胞内部复杂度的侧向散射光。 流式细胞仪将检测的各种光信号转换为电 信号传输给数据处理系统。
步骤 13 ,数据处理系统对流式细胞仪检测的各种数据进行处理，计算各样本 液的差值和比值， 所述比值为落入对照区域的细胞数与全部细胞数的比值， 比值 例如可以是一个百分比， 或与百分比相等的小数。
差值的计算步骤包括：
步骤 131 , 根据第一荧光强度值计算该试验样本液的荧光强度。 在计算样本 液的荧光强度时， 可根据该试验样本液中感兴趣细胞的第一荧光强度值来计算， 感兴趣细胞可以是全部白细胞， 也可以仅是淋巴细胞。 因 HLA- B27抗原尤其在 淋巴细胞表面含量丰富， 因此在一种较佳的实施例中， 可只根据淋巴细胞的第一 荧光强度值计算该试验样本液的荧光强度， 具体步骤包括： 利用步骤 12收集的 散射光， 对白细胞进行分类统计， 识别出淋巴细胞， 获取淋巴细胞的第一荧光强 度值， 根据这些淋巴细胞的第一荧光强度值计算该试验样本液的荧光强度。 荧光 强度可以是这些第一荧光强度值的平均值， 也可以是这些第一荧光强度值的中位 值。
步骤 132, 计算试验样本液的差值。 获取试验样本液的荧光强度与同型对照 数据， 同型对照数据中包括同型对照的荧光强度， 同型对照的荧光强度可以是设 备中预存的值， 也可以是同型对照样本液的荧光强度。 例如， 将试验样本分成两 份， 一份用于制备试验样本液， 另一份用于制备同型对照样本液， 即将荧光检测 试剂的同型对照试剂和另一份试验样本混合处理后得到该试验样本液的同型对 照样本液， 采用流式细胞仪检测同型对照样本液得到同型对照样本液的各细胞的 第一荧光强度值， 根据同型对照样本液中感兴趣细胞的第一荧光强度值， 计算同 型对照的荧光强度。 计算试验样本液的荧光强度与同型对照的荧光强度的差值作 为该试 3 样本液的差值。
比值的计算步骤包括：
步骤 133 , 生成同型对照散点图。 获取同型对照数据， 同型对照数据中包括 同型对照样本液中感兴趣细胞第一荧光强度值和第二荧光强度值， 根据同型对照 样本液中感兴趣细胞的第一荧光强度值和第二荧光强度值形成二维的同型对照 散点图。 该散点图为多个由第一荧光强度值和第二荧光强度值形成的二维数组的 数据集合， 或根据第一荧光强度值和第二荧光强度值形成的一个二维图， 例如以 第一荧光强度值为横坐标， 第二荧光强度值为纵坐标， 反之亦可， 图中的每个点 代表一个细月包。
步骤 134, 在同型对照散点图中设门。 设门是指设定一些条件， 将符合某种 条件的细胞归为一类， 从而将细胞分类。 本步骤中， 通过比较第一荧光强度值的 大小和第二荧光强度值的大小， 使得预定比例的细胞分布在门限的指定区域。 例 如设定一个第一阔值和一个第二阔值， 指定区域为第一荧光强度值小于第一阈值 和第二荧光强度值小于第二阔值的区域， 则第一阈值和一个第二阈值构成门限， 该门限使落入该指定区域的细胞占散点图中全部细胞数的比例应满足预定比例， 即该门限使落入该指定区域的细胞占散点图中全部细胞数的比例应是一个预定 比例， 或在该预定比例可接受的误差范围内， 预定比例可以是一个根据实际要求 预设的一个值， 例如使落入该指定区域的细胞占散点图中全部细胞数的比例为 95%或 98%或 98% ± 0.5%等。
步骤 135 , 生成试验样本散点图。 获取试验样本液中感兴趣细胞的第一荧光 强度值和第二荧光强度值，根据试验样本液中感兴趣细胞的第一荧光强度值和第 二荧光强度值形成试验样本散点图。
步骤 137, 在试验样本散点图中釆用同样门限划分细胞群体， 计算落入指定 区域的对照区域的细胞数占试验样本散点图中全部细胞数的比值。 对照区域为在 沿第一荧光强度值正向上不同于指定区域的区域 , 例如指定区域为第一荧光强度 值小于第一阈值和第二荧光强度值小于第二阈值的区域, 则对照区域为第一荧光 强度值大于第一阈值和第二荧光强度值小于第二阈值的区域。 当设定门限为十字 门时， 指定区域为十字门的左下象限, 对照区域为十字门的右下象限。
步骤 14,将所有的试验样本的阴阳性以及各样本的差值和比值进行统计,根 据试验样本的已知的阴阳性以及可接受的灵敏度和特异性标准 ,从多个试验样本 中确定出一个样本的差值和比值作为判断未知样本阴阳性的荧光临界值和比值 临界值。其中，可接受的灵敏度和特异性标准根据用户的要求和具体设备而确定。
在优选的具体实例中， 在采用流式细胞仪检测试验样本液之前， 先对流式细 胞仪进行荧光值校准，调节流式细胞仪荧光检测通道的电压,将校准荧光微球在荧 光检测通道显示的荧光值调整到校准值。
下面以一个实例进一步说明如何确定未知样本的 HLA- B27阴阳性判断标准。
1、 收集血液样本 348例， 其中阳性样本 182例， 阴性样本 166例， 检测数 据如表 1所示。 将 HLA- B27检测试剂（即用于检测 HLA- B27抗原的荧光检测试 剂）和血液样本混合， 在室温下反应一段时间， 对样本进行染色。
2、 将染色后样本通过流式细胞仪检测。 在检测样本之前对流式细胞仪进行 校准， 调节流式细胞仪第一通道 FL1和第二通道 FL2的通道电压， 根据釆用的 第一荧光物质 FITC和第二荧光物质 PE的参数， 将校准荧光微球在 FITC/PE通 道显示的荧光值调整到校准值。 通过流式细胞仪检测样本时， 通过前向散射光信 号和侧向散射光信号， 可以识别出样本中的淋巴细胞， 收集该淋巴细胞 FL1 及 FL2通道的荧光值信号。
3、 根据各淋巴细胞 FL1通道的荧光值信号， 计算其中位值或平均值， 得到 该样本 FL1通道的荧光强度。
4、 计算各样本的差值 MFI (差值)， 计算公式为：
MFI(差值) = MFI(FITC- HLA- B27)- MFI(FITC- IgG2a)
其中 ， MFI(FITC- HLA- B27)为各样本液 FL1 通道的荧光强度， MFI(FITC-IgG2a)为同型对照样本液的 FL1通道的荧光强度。 5、 通过同型对照设门 , 得到 FITC-HLA-B27/PE-HLA-B7散点图十字门右下 象限细胞占淋巴细胞的百分比 Q4。
6、 统计以上结果， 请参考表 1 , 表中， MFI— IgG2为同型对照的荧光强度， MFI— B27为样本的荧光强度， MFI(差值)为 MFI— B27与 MFI— IgG2的差值， Q4 为 FITC- HLA-B27/PE- HLA- B7散点图十字门右下象限细胞占淋巴细胞的百分比。 经过分析得知， 当 MFI (差值）> 540且 Q4 > 80%时， 检测结果的 £阳性和支阴性 样本总数最低， 检测结果如表 2 所示， 敏感性 =180/182=98.9% , 特异性 =166/166=100%,得到的 MFI(差值)和 Q4值即为阴阳性判定的荧光临界值和比值 临界值。
表 1
MFI_ MFI— MFI( 差 样本的
Q4 %
IgG B27 值） 阴阳性
17 2486 92.9 2469 阳性
19 2230 90 2211 阳性
19 1549 93.9 1530 阳性
17 1374 94.8 1357 阳性
19 1375 97 1356 阳性
17 1320 89.9 1303 阳性
15 1315 89.4 1300 阳性
18 1302 91.8 1284 阳性
19 1274 93.2 1255 阳性
14 1269 15.1 1255 阳性
17 1267 90.6 1250 阳性
17 1267 92.2 1250 阳性
17 1254 90.6 1237 阳性
17 1241 96.3 1224 阳性
18 1231 92.8 1213 阳性
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20 29 1.9 9 阴性
19 27 2.2 8 阴性
18 25 1.5 7 阴性
17 24 0.7 7 阴性
20 26 2.1 6 阴性
14 20 1.4 6 阴性
21 27 1.2 6 阴性
19 24 0.9 5 阴性
21 26 1 5 阴性
18 23 2 5 阴性
17 22 1.6 5 阴性
18 23 1.4 5 阴性
19 24 0.9 5 阴性
17 22 1 5 阴性
19 24 0.6 5 阴性
17 21 0.3 4 阴性
20 23 0.9 3 阴性
20 23 0.9 3 阴性
18 20 1.7 2 阴性
35 37 0.9 2 阴性
17 19 0.3 2 阴性
20 22 0.5 2 阴性
21 22 0.7 1 阴性
14 15 0.5 1 阴性 表 2
Figure imgf000023_0001
阳性 180 0 180 阴性 2 166 168 总计 182 166 348 在一实施方式中， 当采用上述实施例确定的荧光临界值和比值临界值标准判 断未知样本的阴阳性时， 其判断流程如图 3所示， 包括以下步骤：
步骤 21 ,采用样本液制备系统将被测血液样本和荧光检测试剂混合处理，制 成需要的被测样本液。 荧光检测试剂中包括采用第一荧光物质标记的 HLA-B27 抗体和采用第二荧光物质标记的干扰抗体。 HLA-B27 抗体例如可以是单克隆抗 体, 第二荧光物质与第一荧光物质为不同发光波长的荧光物质。 当将荧光检测试 剂和被测样本混合后， 被荧光标记的 HLA-B27抗体和 HLA-B27抗原结合, 被荧 光标记的千扰抗体和千扰抗原 (例如 HLA-B7抗原 )结合， 因此携带有 HLA-B27 抗原和千扰抗原的白细胞被荧光物质染色, 同时被荧光标记的 HLA-B27抗体也 与干扰抗原（例如 HLA- B7抗原）发生交叉反应。 通过清洗分离， 将未与白细胞 结合的标记物清除， 得到染色后的被测样本液。
步骤 22,釆用流式细胞仪检测被测样本液。具体检测步骤可与实施例一中的 检测步骤相同。 经过检测得到被测样本液中被标记细胞的第一荧光强度值和第二 荧光强度值， 根据需要还可检测得到细胞因被光束照射而发出的散射光。 流式细 胞仪将检测的各种光信号转换为电信号传输给数据处理系统。
步骤 23 ,数据处理系统对流式细胞仪检测的各种数据进行处理，计算被测样 本液的差值和比值。
差值的计算步骤包括：
步骤 231 , 根据第一荧光强度值计算该被测样本液的荧光强度。 在计算样本 液的荧光强度时， 可根据该被测样本液中感兴趣细胞的第一荧光强度值来计算。 感兴趣细胞可以是全部白细胞，也可以仅是淋巴细胞。淋巴细胞的识别步骤包括： 利用步骤 22收集的散射光， 对白细胞进行分类统计， 识别出淋巴细胞， 获取淋 巴细胞的第一荧光强度值，根据这些淋巴细胞的第一荧光强度值计算该被测样本 液的荧光强度。 在优选的实施例中， 本步骤中的感兴趣细胞应与判断标准确定过 程中釆用的感兴趣细胞群体一致。 荧光强度可以是这些第一荧光强度值的平均 值， 也可以是这些第一荧光强度值的中位值。 在优选的实施例中, 本步骤中的荧 光强度也应与判断标准确定过程中荧光强度的算法一致。
步骤 232, 计算被测样本液的差值。 获取被测样本液的荧光强度与同型对照 数据， 同型对照数据中包括同型对照的荧光强度, 同型对照的荧光强度可以是设 备中预存的值， 也可以是同型对照样本液的荧光强度。 例如， 将被测样本分成两 份， 一份用于制备被测样本液， 另一份用于制备同型对照样本液， 即将荧光检测 试剂的同型对照试剂和另一份被测样本混合处理后得到该被测样本液的同型对 照样本液， 采用与被测样本液同样的方法用流式细胞仪对同型对照样本液进行检 测， 得到同型对照样本液的荧光强度。 将被测样本液的荧光强度与同型对照的荧 光强度的差值作为该被测样本液的差值。
比值的计算步骤包括：
步骤 233 , 生成同型对照散点图。 获取被测样本的同型对照数据， 同型对照 数据中包括同型对照样本液中感兴趣细胞第一荧光强度值和第二荧光强度值，根 据同型对照样本液中感兴趣细胞的第一荧光强度值和第二荧光强度值形成二维 的同型对照散点图。
步骤 234 , 在同型对照散点图中设定门限， 该门限使落入指定区域的细胞占 散点图中全部细胞数的比例应满足预定比例， 即该门限使落入该指定区域的细胞 占散点图中全部细胞数的比例应是一个预定比例， 或在该预定比例可接受的误差 范围内， 预定比例可以是一个根据实际要求预设的一个值， 例如使落入该指定区 域的细胞占散点图中全部细胞数的比例等于 95%、 98%或其附近的一个值。 判断 步骤 235 , 生成被测样本散点图。 获取被测样本液中感兴趣细胞的第一荧光 强度值和第二荧光强度值，根据被测样本液中感兴趣细胞的第一荧光强度值和第 二荧光强度值形成被测样本散点图。
步骤 237, 在被测样本散点图中釆用同样门限划分细胞群体， 计算落入指定 区域的对照区域的细胞数占被测样本散点图中全部细胞数的比值。 当设定门限为 十字门时， 指定区域为十字门的左下象限， 对照区域为十字门的右下象限。 步骤 24,将差值和比值分别和荧光临界值和比值临界值比较，根据比较结果 判断被测样本针对于 HLA-B27抗原的阴阳性。 该判断可以是操作者根据数据处 理系统输出的差值和比值自行与荧光临界值和比值临界值比较 ,也可以是将荧光 临界值和比值临界值预先输入数据处理系统中， 由数据处理系统在计算得到被测 样本的差值和比值后自动将差值和比值分别和荧光临界值和比值临界值比较。 判 断规则例如可以是： 当被测样本的差值大于或等于荧光临界值， 且该被测样本的 比值大于或等于比值临界值时， 被测样本液为阳性， 否则为阴性。 当然， 本领域 技术人员应该理解， 判断规则中的任一个 "大于或等于" 也可以改为 "大于"。
才艮据上述实施例的判断标准的建立方法和未知样本阴阳性的判断方法， 一方 面由于采用同型对照的荧光强度作为底数， 减少了非特异性结合的影响， 确定了 合理的荧光临界值， 所以减少了非特异性结合对检测结果的千扰， 提高了对 HLA- B27抗原表达检测结果的准确性。 另一方面， 根据设定的比值临界值， B27 的荧光强度大于该临界值， 而 B7的荧光强度小于该临界值， 荧光强度大于该临 界值的应该是真实表达 B27抗原的细胞。 因此根据被测未知样本的 Q4的结果， 可准确的判断出 HLA- B27抗原的表达， 从而排除了 HLA- B7抗原对检测结果的 影响， 显著的减低了检测结果的假阳性。
上述实施例中， 通过对流式细胞仪进行荧光校准， 可以很好的实现不同机器 以及不同流式机型之间的相互移植，检测结果在不同的实验室之间具有很好的可 比性。
下面以两个实例进一步说明利用上述实施例确定的荧光临界值和比值临界 被测样本 1 :
将已知的 HLA- B27阴性的新鲜抗凝血作为被测样本 1和 HLA-B27检测双色 试剂反应， 制备好的样本通过流式细胞法检测， 通过前向散射光 (FSC)和侧向散 射光 (SSC)选定淋巴细胞， 如图 4 所示， 在前向散射光 (FSC)和侧向散射光 (SSC) 形成的散点图中， 左下角 P1 区域中为淋巴细胞。 根据同型对照 （检测中使用的 一种对照试剂， 具体为 FITC- IgG2a/PE- IgGl双色同型对照） 的第一 FL1和第二 FL2的荧光强度值， 设置 FL1/FL2散点图十字门， 如图 5所示， 假设十字门左下 象限为指定区域，要求有 98%的细胞落入左下象限。 当使 99%的细胞的第一荧光 强度值分布在第一阈值分界线左边，且 99%的细胞的第二荧光强度值分布在第二 阈值分界线下边， 第一阈值分界线和第二阈值分界线形成一^卜字门, 该十字门左 下象限中分布的细胞数占散点图中全部细胞数的 98%。 如图 6所示为被测样本 1 的第一 FL 1和第二通道 FL2的荧光强度值形成的散点图， 在被测样本 1 的散点 图中， 采用与同型对照散点图中相同的十字门， 计算被测样本 1的 MFI(差值)值 及 Q4值， Q4值为落入十字门第四象限的细胞数占散点图中全部细胞数的百分比， 第四象限为第三象限的对照象限。检测结果 MFI(差值）值< 540且 Q4 < 80%,检 测结果为阴性。
被测样本 2:
将已知的 HLA- B27阳性的新鲜抗凝血作为被测样本 2和 HLA- B27检测双色 试剂反应，通过前向散射光 (FSC)和侧向散射光 (SSC)选定淋巴细胞，如图 7所示， 在前向散射光 (FSC)和侧向散射光 (SSC)形成的散点图中， 左下角 P1 区域中为淋 巴细胞。根据同型对照（检测中使用的一种对照试剂，具体为 FITC- IgG2a/PE- IgGl 双色同型对照 ) 的第一通道 FL1和第二通道 FL2的荧光强度值， 设置 FL1/FL2 散点图十字门， 如图 8所示， 假设十字门左下象限为指定区域， 同样要求有 98% 的细胞落入左下象限。 则分布在第一阈值分界线左边的细胞数和分布在第二阈值 分界线下边的细胞数仍然都为 99%,第一阔值分界线和第二阈值分界线形成一" 字门， 该十字门左下象限中分布的细胞数占散点图中全部细胞数的 98%。 如图 9 所示为被测样本 2的第一 FL1和第二 FL2的荧光强度值形成的散点图， 在被测 样本 2的散点图中， 采用与同型对照散点图中相同的十字门， 计算被测样本 2的 MFI(差值)值及 Q4值， 检测结果为 MFI(差值）值 > 540且 Q4 > 80%, 检测结果 为阳性。
在一实施方式中， 判断标准包括荧光临界值和比值临界值， 在其它的实施例 中， 判断标准也可以只包括荧光临界值和比值临界值中的任一个， 相应的， 在对 未知样本进行 HLA-B27抗原表达的检测中， 也只须与一个判断标准比较。
上述实施例中， 感兴趣抗原为 HLA- B27抗原， 干扰抗原为 HLA- B7抗原。 在其他的实施例中， 感兴趣抗原也可以为其它细胞表面抗原， 相应的干扰抗原为 该细胞表面抗原有交叉反应的抗原。 上述实施例中， 使用的抗体为单克隆抗体, 在其他实施例中 , 也可以使用多克隆抗体。
本领域技术人员可以理解， 上述实施方式中各种方法的全部或部分步骤可以 通过程序来指令相关硬件完成， 该程序可以存储于一计算机可读存储介质中， 存 储介质可以包括： 只读存储器、 随机存储器、 磁盘或光盘等。 但并不能因此而理解为对本发明专利范围的限制。 应当指出的是， 对于本领域的 普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进， 这些都属于本发明的保护范围。 因此， 本发明专利的保护范围应以所附权利要求 为准。

Claims

权利要求书
1、 一种用于判断未知样本阴阳性的判断标准的确定方法， 其特征在于， 包 括：
将多个试验样本分别和荧光检测试剂混合处理， 得到各自对应的试验样本 液， 所述多个试验样本针对于感兴趣抗原已知被确定为阴性或阳性， 所述荧光检 测试剂中包括采用第一荧光物质标记的与感兴趣抗原对应的抗体；
采用流式细胞仪检测各试验样本液中各细胞发出的第一荧光物质的荧光信 号， 获得第一荧光强度值；
根据所述试验样本液中感兴趣细胞的第一荧光强度值， 计算该试验样本液的 荧光强度；
计算所述试验样本液的荧光强度与同型对照的荧光强度的差值； 及 根据所述试验样本的已知的阴阳性， 从多个试验样本的差值中确定出一个最 合适值作为判断未知样本阴阳性的荧光临界值。
2、 如权利要求 1 所述的方法， 其特征在于， 所述荧光检测试剂中还包括釆 用第二荧光物质标记的与感兴趣抗原的千扰抗原对应的抗体， 所述第二荧光物质 的发光波长与所述第一荧光物质的发光波长不同， 所述方法还包括：
在采用流式细胞仪检测各试验样本液的过程中， 还检测试验样本液中各细胞 发出的第二荧光物质的荧光信号， 获得第二荧光强度值；
在由同型对照样本液的感兴趣细胞的第一荧光强度值和第二荧光强度值形 成的同型对照散点图中， 设定门限， 使预定比例的细胞分布在所述门限的指定区 域；
在由所述试验样本液中感兴趣细胞的第一荧光强度值和第二荧光强度值形 成的试验样本散点图中， 计算落入指定区域的对照区域的细胞数占试验样本散点 图中全部细胞数的比值； 及
根据试验样本的已知的阴阳性 , 从多个试验样本的比值中确定出一个最合适 值作为判断未知样本阴阳性的比值临界值。
3、 如权利要求 1 所述的方法， 其特征在于， 所述同型对照为荧光检测试剂 的同型对照试剂与试险样本混合处理后所得的样本液， 所述同型对照样本液和试 验样本液采用同一试验样本制得， 釆用流式细胞仪检测同型对照样本液得到同型 对照样本液的各细胞的第一荧光强度值 ,根据同型对照样本液中感兴趣细胞的第 一荧光强度值, 计算同型对照的荧光强度。
4、 如权利要求 2所述方法， 其特征在于， 所述同型对照为荧光检测试剂的 同型对照试剂与试验样本混合处理后所得的样本液, 所述同型对照样本液和试-险 样本液采用同一试验样本制得， 采用流式细胞仪检测同型对照样本液得到同型对 照样本液的各细胞的第一荧光强度值和第二荧光强度值。
5、 一种提高细胞表面抗原表达检测准确性的方法， 其特征在于包括： 将被测样本与荧光检测试剂混合处理， 得到染色后的被测样本液, 荧光检测 试剂中包括采用第一荧光物质标记的与感兴趣抗原对应的抗体；
采用流式细胞仪检测被测样本液， 检测被测样本液中各细胞发出的第一荧光 物质的荧光信号， 获得第一荧光强度值；
根据被测样本液中感兴趣细胞的第一荧光强度值计算被测样本液的荧光强 度; 及
计算被测样本液的荧光强度与同型对照的荧光强度的差值， 所述差值用于与 预设的荧光临界值进行比较。
6、 如权利要求 5所述的方法， 其特征在于， 所述荧光临界值通过权利要求 1 所述的方法获得。
7、 如权利要求 5所述的方法， 其特征在于， 还包括：
将所述差值与荧光临界值进行比较， 根据比较结果输出被测样本液阴阳性的 判断结果； 当所述差值大于或等于荧光临界值，被测样本液为阳性， 否则为阴性。
8、 如权利要求 5 所述的方法， 其特征在于， 所述荧光检测试剂中还包括采 用第二荧光物质标记的抗体， 所述抗体为与感兴趣抗原的干扰抗原对应的抗体， 第二荧光物质的发光波长与第一荧光物质的发光波长不同， 所述方法还包括： 在采用流式细胞仪检测被测样本液的过程中， 还检测被测样本液中各细胞发 出的第二荧光强度值；
在由同型对照样本液的感兴趣细胞的第一荧光强度值和第二荧光强度值形 成的同型对照散点图中， 设定门限， 使预定比例的细胞分布在门限的指定区域； 及
在由被测样本液中感兴趣细胞的第一荧光强度值和第二荧光强度值形成的 被测样本散点图中, 计算落入指定区域的对照区域的细胞数占被测样本散点图中 全部细胞数的比值； 所述比值用于与预设的比值临界值进行比较。
9、 如权利要求 8所述的方法, 其特征在于， 所述比值临界值通过权利要求 2 所述的方法获得。
10、 如权利要求 8所述的方法， 其特征在于， 还包括：
将所述差值与荧光临界值进行比较， 将所述比值与比值临界值进行比较; 及 根据比较结果输出被测样本液阴阳性的判断结果； 当所述差值大于或等于荧 光临界值， 且所述比值大于或等于比值临界值时， 被测样本液为阳性, 否则为阴 性。
11、 如权利要求 1或 5所述的方法， 其特征在于， 在釆用流式细胞仪检测各 试-睑样本液之前对流式细胞仪进行荧光值校准。
12、如权利要求 1或 5所述的方法，其特征在于，所述感兴趣抗原为 HLA- B27。
13、 如权利要求 12 所述的方法， 其特征在于， 第一荧光标记的抗体为 HLA-B27单克隆抗体 , 和 /或干扰抗原为 HLA-B7 , 和 /或第二荧光标记的抗体为 HLA-B7单克隆抗体。
14、 如权利要求 1或 5所述的方法， 其特征在于， 在釆用流式细胞仪检测各 样本液的过程中， 还检测样本液中细胞被光束照射后发出的散射光, 根据散射光 统计样本液中的淋巴细胞， 在计算样本液的荧光强度时将淋巴细胞作为感兴趣细 胞， 根据统计出的样本液中淋巴细胞的第一荧光强度值计算荧光强度。
15、 如权利要求 2或 8所述的方法， 其特征在于， 所述门限为十字门， 所述 指定区域为十字门的左下象限， 对照区域为十字门的右下象限。
16、 一种用于检测细胞表面抗原表达的设备， 其特征在于， 包括:
样本液制备系统， 用于将被测体液样本与荧光检测试剂混合处理， 得到染色 后的被测样本液， 荧光检测试剂中包括采用第一荧光物质标记的与感兴趣抗原对 应的抗体；
流式细胞仪， 用于检测被测样本液， 所述流式细胞仪检测被测样本液中各细 胞发出的第一荧光物质的荧光信号， 获得第一荧光强度值； 及
数据处理系统， 用于接收流式细胞仪输出的数据， 根据被测样本液中感兴趣 细胞的第一荧光强度值计算被测样本液的荧光强度, 计算被测样本液的荧光强度 与同型对照的荧光强度的差值， 所述差值用于与预设的荧光临界值进行比较。
17、 如权利要求 16所述的设备， 其特征在于， 所述荧光临界值通过权利要 求 1所述的方法获得。
1 8、 如权利要求 16所述的设备， 其特征在于， 所述流式细胞仪在采用流式 细胞仪检测各被测样本液的过程中 ,还检测样本液中细胞被光束照射后发出的散 射光， 数据处理系统根据流式细胞仪检测的散射光统计样本液中的淋巴细胞, 计 算被测样本液的荧光强度时将淋巴细胞作为感兴趣细胞, 根据统计出的样本液中 淋巴细胞的第一荧光强度值计算荧光强度。
19、 如权利要求 16所述的设备， 其特征在于， 所述荧光检测试剂中还包括 采用第二荧光物质标记的与感兴趣抗原的干扰抗原对应的抗体， 第二荧光物质发 光波长与第一荧光物质的发光波长不同， 所述流式细胞仪在检测被测样本液的过 程中， 还检测被测样本液中各细胞发出的第二荧光物质的荧光信号， 获得第二荧 光强度值； 所述数据处理系统根据同型对照样本液中感兴趣细胞的第一荧光强度 值和第二荧光强度值形成二维的同型对照散点图 , 在同型对照散点图中设定门 限， 使预定比例的细胞分布在门限的指定区域; 根据被测样本液中感兴趣细胞的 第一荧光强度值和第二荧光强度值形成被测样本散点图， 计算落入指定区域的对 照区域的细胞数占被测样本散点图中全部细胞数的比值； 所述比值用于与预设的 比值临界值进行比较。
20、 如权利要求 19所述的设备， 其特征在于， 所述比值临界值通过权利要 求 2所述的方法获得。
21、 如权利要求 19所述的设备， 其特征在于， 所述门限为十字门， 所述指 定区域为十字门的左下象限， 对照区域为十字门的右下象限。
22、 如权利要求 19所述的设备， 其特征在于，
数据处理系统将所述差值与荧光临界值进行比较， 将所述比值与比值临界值 进行比较， 并根据比较结果输出被测样本液阴阳性的判断结果。
23、 如权利要求 22所述的设备， 其特征在于， 当所述差值大于或等于荧光 界值，且所述比值大于或等于比值临界值时，被测样本液为阳性， 否则为阴性。
24、 如权利要求 16所述的设备， 其特征在于， 所述感兴趣抗原为 HLA- B27。
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