CN112161913A - Analysis method and equipment for flow type fluorescence analysis system - Google Patents
Analysis method and equipment for flow type fluorescence analysis system Download PDFInfo
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- CN112161913A CN112161913A CN202011027010.6A CN202011027010A CN112161913A CN 112161913 A CN112161913 A CN 112161913A CN 202011027010 A CN202011027010 A CN 202011027010A CN 112161913 A CN112161913 A CN 112161913A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
Abstract
The invention discloses an analysis method and equipment for a flow-type fluorescence analysis system, which comprises the steps of establishing an immunoassay standard concentration curve and calculating the concentration of a test sample: according to the flow type fluorescence data acquisition, providing a standard curve template of a corresponding detection item, and completing standard sample data acquisition; in the standard curve template of the corresponding detection item, all samples collected by the corresponding detection item are displayed in the template, and one sample of a plurality of selected samples is automatically analyzed to generate a standard curve; and obtaining the concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve. The invention sets two steps, establishment of an immunity detection standard concentration curve and automatic calculation of the concentration of the test sample, fluorescence values of the standard sample and the test sample can be easily obtained through self-animation gate analysis, manual animation gate analysis can be adopted if automatic gate drawing fails, the operation is convenient, the compatibility is strong, and the application range is wide.
Description
Technical Field
The invention relates to the field of flow type fluorescence analysis in medical treatment, in particular to an analysis method and equipment for a flow type fluorescence analysis system.
Background
Conventional flow cytometers (including hematology analyzers) are instruments that can rapidly quantify the physical or chemical properties of a cell population and accurately sort cells based on these characteristic signal differences. The flow cytometer detects the scattered light signal and fluorescence excitation signal of each cell after being irradiated by the laser to reflect the physical characteristics and biochemical characteristics of the cell, such as the size, activity, granularity, number of nucleic acid and expression of antigen molecules.
The flow-type fluorescence detection technology is an immunoassay technology based on the detection principle of a flow cytometer, and uses microspheres to replace cells, and uses fluorescence generated after antigen-antibody specific binding as a signal to detect a specific substance in a sample. This technique is similar to chemiluminescence and is used to capture a target protein (or polypeptide) in a sample by coating a capture antibody on a microsphere and then calculating the concentration of the target protein using the fluorescence intensity produced by a fluorescently labeled detection antibody. In recent years, the flow-type fluorescence detection technique has been widely used clinically. For example, the double antibody sandwich method is used for measuring tumor markers, cytokines and pathogen antibodies of macromolecules, and the competition method is used for detecting hormone and drug concentration of small molecules, so that the detection of related items can provide powerful basis for diagnosis, treatment and prognosis of clinical diseases.
The flow-type fluorescence detection technology relates to two core technologies of fluorescence coding microspheres and flow-type fluorescence detection. The fluorescence coding microsphere technology is characterized in that one or more kinds of fluorescein or quantum dots with different concentrations are doped into microspheres, so that the microspheres have fluorescence with different intensities and colors, and a series of coding microsphere arrays are formed. A plurality of different fluorescence coding microspheres are added into the same test tube to react with the sample, and each microsphere is only combined with a specific substance to be detected and rarely interferes with each other. The separated detection object is detected on a flow cytometer, and each microsphere can be independently used for measuring one index, so that high-throughput joint detection of several or even dozens of projects can be realized in one test tube.
Flow-based fluorescence detection systems are mainly divided into two subsystems, namely hardware and data analysis, and the analysis subsystem usually comprises three parts: data acquisition, standard curve establishment and concentration point calculation. However, the conventional flow cytometer generally only provides conventional statistics on the percentage, number, and emission intensity of particles, and the like, and cannot calculate a standard curve and also cannot provide concentration statistics of detection items. For example, the current flow cytometry method using flow fluorescence method for immune detection requires a set of software (such as FCAP) specially used for analyzing the data of flow fluorescence immune detection, besides a set of traditional data acquisition software, and all flow fluorescence analysis software cannot realize automatic analysis.
Therefore, the existing method is to collect data on the acquisition software matched with the flow cytometer, then to export the data in a specific format, then to import the exported data into the analysis software of the flow fluoroimmunoassay, to perform operations such as manual gate analysis, and finally to export results, and the data acquisition and the data analysis are two independent systems. Among the drawbacks are: 1. two independent software are needed, and the operation of exporting and importing is complicated; 2. the streaming data formats have various versions, the compatibility of data is problematic, and the data of some instruments can not be imported into special analysis software for the streaming fluorescence immunoassay; 3. the flow type fluorescence immunoassay reagent has a plurality of types, and flow type fluorescence immunoassay analysis software cannot cover all types and can only aim at the related reagent of a specific company; 4. needs analysis operations such as manual animation door and standard curve establishment, and has higher technical requirements on operators.
Accordingly, the prior art is deficient and needs improvement.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the analysis method for the flow-type fluorescence analysis system is simple to operate, high in compatibility and wide in application range.
The technical scheme of the invention is as follows: an analysis method for a flow-type fluorescence analysis system, comprising the steps of:
s1, establishing an immunity detection standard concentration curve;
wherein, step S1 further includes the steps of:
s11, collecting standard sample data: according to the flow type fluorescence data acquisition, providing a standard curve template of a corresponding detection item, and completing standard sample data acquisition;
s12, standard curve generation: in the standard curve template of the corresponding detection item, all samples collected by the corresponding detection item are displayed in the template, and one sample of a plurality of selected samples is automatically analyzed to generate a standard curve;
s2, automatically calculating the concentration of the test sample: and obtaining a concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve in the step S1.
With the above technical solution, in the analyzing method for the flow-type fluorescence analyzing system, the step S11 further includes the following steps:
s111, reading data: reading data acquired by the selected standard sample in the sample data acquisition module;
s112, automatic door drawing analysis or hand moving door analysis:
the self-animation gate analysis comprises the steps of circling main cliques on scatter diagrams generated by a front scatter FSC channel and a side scatter SSC channel, and circling corresponding code cliques on the code channel scatter diagrams, wherein the number of the cliques on the code channel scatter diagrams and the number of the cliques on the code channel scatter diagrams are determined by corresponding immunodetection reagents, data of a test channel in a gate are obtained, the number of bits is calculated to be the fluorescence value of the test channel, and the fluorescence value is automatically input into an analysis software system;
hand motion gate analysis the system calculates the statistical values after the gate is drawn by flow fluorescence analysis hand motion gate, and then the calculated fluorescence values are manually collected into the analysis software system.
With the above technical solutions, the analyzing method for the flow-type fluorescence analyzing system further includes, in step S12, the following steps:
s121, curve fitting: generating concentration gradient points according to the highest concentration and concentration gradient preset by the system, and performing curve fitting on the concentration gradient points and fluorescence values obtained by gating analysis, wherein the x axis is the fluorescence value, and the y axis is the concentration gradient points;
s122, saving and displaying: and displaying the fitting result in the graph, and simultaneously storing the fitting result in a computer to facilitate the calling when the test sample data is calculated.
With the above technical solutions, in the analysis method for the flow fluorescence analysis system, in step S2, test sample data is obtained on a test sample interface, and a fluorescence value of the test sample is obtained through self-animation gate analysis or hand-animation gate analysis.
With the above technical solutions, in the analysis method for the flow-type fluorescence analysis system, in step S111, the standard sample includes at least one test tube, and if there are a plurality of test tubes, data of each test tube is read respectively.
By adopting the technical scheme, in the analysis method for the flow-type fluorescence analysis system, the automatic drawing gates of the front scattering FSC channel and the side scattering SSC channel and the automatic drawing gate of the coding channel adopt a processing method based on images.
By adopting the technical scheme, in the analysis method for the flow type fluorescence analysis system, the image-based processing method is an image morphology method or an image segmentation algorithm.
By adopting the technical scheme, the invention is provided with two steps, the establishment of the immunity detection standard concentration curve and the automatic calculation of the concentration of the test sample, the fluorescence values of the standard sample and the test sample can be easily obtained through the self-animation gate analysis, and the manual animation gate analysis can be adopted if the automatic gate drawing fails, so that the operation is convenient, the compatibility is strong, and the application range is wide.
Drawings
FIG. 1 is a schematic diagram of the main steps of the present invention;
FIG. 2 is a detailed view of the main steps of the present invention;
FIG. 3 is a graph of the steps of the immunoassay standard curve of the present invention;
FIG. 4 is a schematic view of the automatic door of the present invention;
FIG. 5 is a graph illustrating the concentration calculation steps of the test sample according to the present invention;
FIG. 6 is a schematic diagram of a standard curve creation embodiment of the present invention;
FIG. 7 is an enlarged view of the left side of FIG. 6;
FIG. 8 is an enlarged view of the right side of FIG. 6;
FIG. 9 is a schematic view of an embodiment of the present invention for testing the concentration of a sample.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
Referring to fig. 1 and fig. 2, the present invention provides an analysis method for a flow-type fluorescence analysis system, which mainly integrates a flow-type fluorescence immunoassay automatic analysis method into conventional data acquisition software to form a complete flow-type fluorescence analysis system. The invention mainly relates to two aspects: establishing an immunity detection standard concentration curve and automatically calculating the concentration of the test sample. The concentration of the test sample can be antigen concentration, protein concentration or cytokine concentration, etc., and the invention is explained by the cytokine concentration, and the test principle of the antigen concentration and the protein concentration is the same, which is not described herein for a long time.
Step S1 is established for the immunoassay standard concentration curve, and mainly includes two parts:
s11, collecting standard sample data: and providing a standard curve template of a corresponding detection item according to the flow type fluorescence data acquisition, and finishing the standard sample data acquisition. The standard sample may contain one or more test tube data depending on the reagent concentration gradient setting of the relevant test item.
S12, standard curve generation: and in the standard curve template of the corresponding detection item, all samples collected by the corresponding detection item are displayed in the template, and when one sample of a plurality of samples is selected, the standard curve is automatically analyzed and generated.
Step S2 is the automatic calculation of test sample concentration: and obtaining a concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve in the step S1.
Preferably, as shown in fig. 3 and 4, the specific process of establishing the standard curve in step S12 is as follows:
s121, reading data: and reading the data acquired by the selected standard sample in the sample data acquisition module. The standard sample comprises at least one test tube, and if a plurality of test tubes exist, the data of each test tube are read respectively. The reading data is the data acquisition method in the prior art flow fluorescence analysis.
S122, automatic door drawing analysis or hand moving door analysis: two door drawing modes are set in the invention, the door drawing mode which is preferably considered is automatic door drawing, and the manual door drawing is considered only when the automatic door drawing is unsuccessful or a user is not satisfied with the automatic door drawing.
The self-animated gate analysis comprises marking main blobs on scatter diagrams generated by a front scatter FSC channel and a side scatter SSC channel, and marking corresponding code blobs on the code channel scatter diagrams, wherein the number of the blobs on the code channel scatter diagrams and the code channel scatter diagrams is determined by corresponding immunodetection reagents, acquiring data of a test channel in a gate, calculating the number of bits as the fluorescence value of the test channel, and automatically inputting the fluorescence value into an analysis software system.
The automatic drawing gates of the front scattering FSC channel and the side scattering SSC channel and the automatic drawing gates of the coding channel adopt an image-based processing method, such as an image morphology method or an image segmentation algorithm, such as a watershed algorithm and the like.
Hand motion gate analysis the system calculates the statistical values after the gate is drawn by flow fluorescence analysis hand motion gate, and then the calculated fluorescence values are manually collected into the analysis software system.
Preferably, as shown in fig. 3 and 4, the specific process of establishing the standard curve in step S12 further includes:
s123, curve fitting: and generating concentration gradient points according to the highest concentration and the concentration gradient preset by the system, and performing curve fitting on the concentration gradient points and the fluorescence values obtained by the gating analysis, wherein the x axis is the fluorescence value, and the y axis is the concentration gradient points. Besides the common four-parameter fitting and five-parameter fitting, the adopted fitting formula can also be customized by a user. The fitting algorithm may employ a commonly used least squares algorithm and its modifications, such as the Levenberg-Marquardt algorithm, etc.
S124, saving and displaying: and displaying the fitting result in the graph, and simultaneously storing the fitting result in a computer to facilitate the calling when the test sample data is calculated.
Preferably, as shown in fig. 5, in step S2, the specific process for calculating the cytokine concentration of the test sample is as follows: and obtaining test sample data on a test sample interface, and obtaining the fluorescence value of the test sample through self-animation gate analysis or hand-animation gate analysis. In the process, automatic gate analysis or manual gate analysis is the same as the method adopted for establishing the standard curve, each group of cell factors are circled, and then the fluorescence value of each group of cell factors is calculated.
According to the standard curve, the x axis is a fluorescence value, and the y axis is a concentration gradient point, so that when the fluorescence value of the cytokine of the test sample is obtained, the fluorescence value is substituted into the standard curve, and the corresponding concentration value of the cytokine can be obtained.
A flow-type fluorescence analysis device is used for the flow-type fluorescence analysis method and comprises an immunoassay standard concentration curve establishing module and a test sample concentration automatic calculation module:
the immunoassay standard concentration curve establishing module comprises: the standard sample data acquisition module and the standard curve generation module; the standard sample data acquisition module provides a standard curve template of a corresponding detection item according to the flow type fluorescence data acquisition to finish the standard sample data acquisition; the standard curve generation module is used for displaying all samples collected by corresponding detection items in a standard curve template of the corresponding detection items, and automatically analyzing and generating a standard curve when one sample of a plurality of samples is selected;
the test sample concentration automatic calculation module: and obtaining a concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve in the step S1.
Furthermore, the device also comprises a liquid path, an optical sensor, a memory and a processor;
a fluid path configured to receive a test sample;
an optical sensor configured to lase a test sample in the fluid path and collect optical feedback from the test sample to provide flow-through fluorescence data acquisition;
a memory storing computer executable instructions that when executed perform an automatic analysis to obtain a test sample concentration value;
a processor configured, upon execution of the computer-executable instructions, to perform the flow fluorescence analysis method described above.
Two examples are provided for further illustration by the above method steps and apparatus.
Example one
As shown in FIGS. 6-8, a single module is provided in a flow-fluorescence assay system for standard curve analysis of immunoassay items (e.g., cytokine assays). In each set of fitting curves, the left list is the cytokine curve sample data collected by the user in real time, and the sample contains a plurality of concentration points and a plurality of cytokine test data according to the requirement of reagent concentration gradient. And the user clicks the sample, the system automatically analyzes the sample data, performs self-moving image gate analysis operation, calculates the fluorescence value of each concentration point of each cytokine group, and displays the fluorescence value in the fluorescence value frame of each cytokine on the right side. And then curve fitting is carried out on the concentration value and the fluorescence value of each cytokine, fitting parameters, errors, curves and the like are obtained according to a fitting method selected by a user, the fitting parameters, the errors, the curves and the like are displayed on an interface, the user can judge whether to adopt the set of results according to the fitting result, if the result is not satisfactory, the fluorescence value obtained by self-gating analysis can be manually input in a fluorescence value frame, and point fitting is carried out to obtain a corresponding fitting result and a corresponding curve.
The system will automatically save the standard curve generated by the sample selected by the current user as the subsequent test sample. The user can decide which set of standard curves to use by clicking on the left sample.
Example two
As shown in fig. 9, when the user tests a certain sample (e.g., cytokine sample), the analysis system automatically circles each cytokine group through an automatic algorithm, and automatically calculates concentration values of each cytokine according to the stored curves, and displays the results in the report. Meanwhile, the user can manually adjust each door, and the concentration result is correspondingly changed to obtain the result desired by the user.
By adopting the technical scheme, the invention is provided with two steps, the establishment of the immunity detection standard concentration curve and the automatic calculation of the concentration of the test sample, the fluorescence values of the standard sample and the test sample can be easily obtained through the self-animation gate analysis, and the manual animation gate analysis can be adopted if the automatic gate drawing fails, so that the operation is convenient, the compatibility is strong, and the application range is wide.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An analysis method for a flow-type fluorescence analysis system, comprising the steps of:
s1, establishing an immunity detection standard concentration curve;
wherein, step S1 further includes the steps of:
s11, collecting standard sample data: according to the flow type fluorescence data acquisition, providing a standard curve template of a corresponding detection item, and completing standard sample data acquisition;
s12, standard curve generation: in the standard curve template of the corresponding detection item, all samples collected by the corresponding detection item are displayed in the template, and one sample of a plurality of selected samples is automatically analyzed to generate a standard curve;
s2, automatically calculating the concentration of the test sample: and obtaining a concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve in the step S1.
2. The analysis method for the flow fluorescence analysis system according to claim 1, wherein the step S12 further comprises the steps of:
s121, reading data: reading data acquired by the selected standard sample in the sample data acquisition module;
s122, automatic door drawing analysis or hand moving door analysis:
the self-animation gate analysis comprises the steps of marking a main cluster on a scatter diagram generated by a front scatter FSC channel and a side scatter SSC channel, and marking a corresponding code cluster on a code channel scatter diagram, wherein the number of the clusters on the code channel scatter diagram and the code channel scatter diagram is determined by corresponding immunodetection reagents, acquiring data of a test channel in a gate, calculating the number of bits as the fluorescence value of the test channel, and automatically inputting the fluorescence value into an analysis software system;
hand motion gate analysis the system calculates the statistical values after the gate is drawn by flow fluorescence analysis hand motion gate, and then the calculated fluorescence values are manually collected into the analysis software system.
3. The analytical method for a flow fluorescence analysis system according to claim 2,
in step S12, the method further includes the steps of:
s123, curve fitting: generating concentration gradient points according to the highest concentration and concentration gradient preset by the system, and performing curve fitting on the concentration gradient points and fluorescence values obtained by gating analysis, wherein the x axis is the fluorescence value, and the y axis is the concentration gradient points;
s124, saving and displaying: and displaying the fitting result in the graph, and simultaneously storing the fitting result in a computer to facilitate the calling when the test sample data is calculated.
4. The analysis method for the flow fluorescence analysis system according to claim 3, wherein in step S2, the test sample data is obtained at the test sample interface, and the fluorescence value of the test sample is obtained by a self-animation gate analysis or a hand-animation gate analysis.
5. The analytical method for a flow fluorescence analysis system according to claim 2,
in step S11, the standard sample includes at least one test tube, and if there are a plurality of test tubes, the data of each test tube is read.
6. The analytical method for a flow fluorometric analytical system of claim 5, wherein the automatic gating of the front scatter FSC channel and the side scatter SSC channel and the automatic gating of the code channel employ an image-based processing method.
7. The analysis method for a streaming fluorescence analysis system of claim 6, wherein the image-based processing method is an image morphology method or an image segmentation algorithm.
8. A flow-type fluorescence analysis device, which is used in the flow-type fluorescence analysis method of any claim 1 to 7, comprising an immunoassay standard concentration curve establishing module and a test sample concentration automatic calculating module;
the immunoassay standard concentration curve establishing module comprises: the standard sample data acquisition module and the standard curve generation module; the standard sample data acquisition module provides a standard curve template of a corresponding detection item according to the flow type fluorescence data acquisition to finish the standard sample data acquisition; the standard curve generation module is used for displaying all samples collected by corresponding detection items in a standard curve template of the corresponding detection items, and automatically analyzing and generating a standard curve when one sample of a plurality of samples is selected;
the test sample concentration automatic calculation module: and obtaining a concentration value of the test sample according to the fluorescence value of the concentration of the test sample and the standard curve in the step S1.
9. A flow fluorometric analytical device according to claim 8, further comprising a fluid path, an optical sensor, a memory, and a processor;
a fluid path configured to receive a test sample;
an optical sensor configured to lase a test sample in the fluid path and collect optical feedback from the test sample to provide flow-through fluorescence data acquisition;
a memory storing computer executable instructions that when executed perform an automatic analysis to obtain a test sample concentration value;
a processor configured to perform the analysis method of any one of claims 1 to 7 when executing the computer-executable instructions.
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