CN110823789A - Method for measuring CD64 of neutrophil granulocytes for clinical infectious diseases - Google Patents
Method for measuring CD64 of neutrophil granulocytes for clinical infectious diseases Download PDFInfo
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- CN110823789A CN110823789A CN201911186780.2A CN201911186780A CN110823789A CN 110823789 A CN110823789 A CN 110823789A CN 201911186780 A CN201911186780 A CN 201911186780A CN 110823789 A CN110823789 A CN 110823789A
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- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 title claims abstract description 54
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 title claims abstract description 54
- 210000000440 neutrophil Anatomy 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 21
- 208000035473 Communicable disease Diseases 0.000 title claims abstract description 14
- 210000001616 monocyte Anatomy 0.000 claims abstract description 14
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000004364 calculation method Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 15
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 238000012545 processing Methods 0.000 claims description 5
- 208000015181 infectious disease Diseases 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 230000010100 anticoagulation Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 210000005087 mononuclear cell Anatomy 0.000 abstract description 3
- 239000013642 negative control Substances 0.000 abstract description 3
- 239000013641 positive control Substances 0.000 abstract description 3
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000009490 IgG Receptors Human genes 0.000 description 1
- 108010073807 IgG Receptors Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 238000012313 Kruskal-Wallis test Methods 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001422 normality test Methods 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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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
- G01N15/1434—Optical arrangements
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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
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention relates to the technical field of neutrophil determination, in particular to a neutrophil CD64 determination method for clinical infectious diseases. The method comprises the following steps: collecting a sample; flow detection; CD64 index calculation: the formula is as follows: the CD64 index ═ (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity)/[ monocyte CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity) - (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity ]. In the method for measuring the neutrophil CD64 for clinical infectious diseases, the fluorescence intensity of the mononuclear cells is used as a positive control, the fluorescence intensity of the lymphocytes is used as a negative control, so that the influence of factors such as instruments, reagents, voltage and the like on the result can be eliminated, and the standardization of the result is facilitated.
Description
Technical Field
The invention relates to the technical field of neutrophil determination, in particular to a neutrophil CD64 determination method for clinical infectious diseases.
Background
CD64 is also known as Fc-gamma receptor protein 1 (FcyRI). CD64 is expressed on the surface of peripheral blood neutrophils in low level, and when the organism is infected, the stimulating factors such as bacterial cell wall lipopolysaccharide, granulocyte colony stimulating factor and interferon can cause CD64 on the surface of neutrophils to be rapidly converted from low level expression to high level expression, and activate neutrophils, so that the CD64 is used as an infection indicator. The activation degree caused by different pathogens has difference, and researches find that the different pathogens have certain discrimination capability on bacterial and viral infection. The determination of CD64 is of great significance for the diagnosis of clinical infectious diseases, but because the determination of fluorescence intensity is affected by instruments, reagents, voltages, etc., it cannot be standardized, and has not been widely used clinically.
Disclosure of Invention
The present invention is directed to a method for measuring neutrophil CD64 in clinical infectious diseases, which solves the problems of the background art mentioned above.
In order to achieve the above object, the present invention provides a method for measuring neutrophil CD64 for clinical infectious diseases, which comprises the following steps:
s1, sample collection: collecting 1-2mL of fasting venous blood, and adopting ethylenediamine tetraacetic acid for anticoagulation;
s2, flow detection: processing a sample, detecting the processed sample blood by adopting a flow cytometer, and obtaining the fluorescence intensity of lymphocytes, monocytes and neutrophils;
s3, CD64 index calculation: the formula is as follows: the CD64 index ═ (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity)/[ monocyte CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity) - (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity ].
Preferably, in the sampling of S1, the fasting venous blood is collected on the same day.
Preferably, in the S2 flow assay, the sample processing method includes the following steps:
s2.1.1, respectively adding 20 μ L of CD64-PE monoclonal fluorescent antibody and 50 μ L of anticoagulated whole blood into the numbered test tubes, and mixing well;
s2.1.2 incubating at room temperature in dark for 15 min;
s2.1.3, adding 1mL of FACSLysin into the test tube, and uniformly mixing;
s2.1.4 incubating at room temperature in dark for 10 min;
s2.1.5, washing cells 2 times with 2mL of PBS buffer;
s2.1.6, cells were suspended in 450. mu.L PBS buffer.
Preferably, in the S2 flow assay, the method for obtaining fluorescence intensity of lymphocytes, monocytes and neutrophils comprises the following steps:
s2.2.1, delineating lymphocytes, monocytes and neutrophils in an FSC/SSC scattergram, respectively;
s2.2.2, respectively establishing a histogram and displaying the geometric mean fluorescence intensity of the three groups of cells;
s2.2.3 10000 cells were taken per sample, and mean fluorescence intensity data for CD64 was obtained for each cell.
Compared with the prior art, the invention has the beneficial effects that: in the method for measuring the neutrophil CD64 for clinical infectious diseases, the fluorescence intensity of the mononuclear cells is used as a positive control, the fluorescence intensity of the lymphocytes is used as a negative control, so that the influence of factors such as instruments, reagents, voltage and the like on the result can be eliminated, and the standardization of the result is facilitated.
Drawings
FIG. 1 is a block diagram of the overall process flow of the present invention;
FIG. 2 is a block flow diagram of a sample processing method of the present invention;
FIG. 3 is a flow chart of the method for obtaining fluorescence intensity of lymphocytes, monocytes and neutrophils according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-3, the present invention provides a technical solution:
the invention provides a method for measuring neutrophil CD64 for clinical infectious diseases, which comprises the following steps:
s1, sample collection: collecting 1-2mL of fasting venous blood, and adopting ethylenediamine tetraacetic acid for anticoagulation;
s2, flow detection: processing a sample, detecting the processed sample blood by adopting a flow cytometer, and obtaining the fluorescence intensity of lymphocytes, monocytes and neutrophils;
s3, CD64 index calculation: the formula is as follows: the CD64 index ═ (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity)/[ monocyte CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity) - (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity ]. Performing normality test on the data to obtain data conforming to normal
One-way ANOVA analysis was performed, and data not conforming to normal distribution was expressed using median (quartile) [ M (P)25,P75)]Described, a nonparametric test (Kruskal-Wallis test) and pairwise comparisons (Mann-Whitney test) were performed to test for differences between groups. Categorizing variables by [ n (%)]Denotes, using x2And (6) checking. P < 0.05 is statistically significant.
In this embodiment, in the sample sampling of S1, the fasting venous blood is collected on the same day.
Further, in the S2 flow test, the method for processing the sample includes the following steps:
s2.1.1, respectively adding 20 μ L of CD64-PE monoclonal fluorescent antibody and 50 μ L of anticoagulated whole blood into the numbered test tubes, and mixing well;
s2.1.2 incubating at room temperature in dark for 15 min;
s2.1.3, adding 1mL of FACSLysin into the test tube, and uniformly mixing;
s2.1.4 incubating at room temperature in dark for 10 min;
s2.1.5, washing cells 2 times with 2mL of PBS buffer;
s2.1.6, cells were suspended in 450. mu.L PBS buffer.
In S2 flow detection, the method for obtaining fluorescence intensity of lymphocytes, monocytes and neutrophils comprises the following steps:
s2.2.1, delineating lymphocytes, monocytes and neutrophils in an FSC/SSC scattergram, respectively;
s2.2.2, respectively establishing a histogram and displaying the geometric mean fluorescence intensity of the three groups of cells;
s2.2.3 10000 cells were taken per sample, and mean fluorescence intensity data for CD64 was obtained for each cell.
The fluorescence intensity of the mononuclear cells is used as a positive control, the fluorescence intensity of the lymphocytes is used as a negative control, the influence of factors such as instruments, reagents, voltage and the like on the result can be eliminated, the test result is stable on different flow cytometers, and no significant difference exists through statistical analysis. And 126 normal persons are detected, and a normal reference value is established. The CD64 measurement is standardized, the influence of different instruments, different reagents and different operators is eliminated, and the fluctuation influence of the instrument voltage can be eliminated.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A method for determining CD64 in neutrophils for clinical infectious diseases, comprising the steps of:
s1, sample collection: collecting 1-2mL of fasting venous blood, and adopting ethylenediamine tetraacetic acid for anticoagulation;
s2, flow detection: processing a sample, detecting the processed sample blood by adopting a flow cytometer, and obtaining the fluorescence intensity of lymphocytes, monocytes and neutrophils;
s3, CD64 index calculation: the formula is as follows: the CD64 index ═ (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity)/[ monocyte CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity) - (neutrophil CD64 fluorescence intensity-lymphocyte CD64 fluorescence intensity ].
2. The method of determining neutrophil CD64 for clinical infectious disease according to claim 1, characterized in that: in the sampling of S1, the fasting venous blood is collected on the same day.
3. The method of determining neutrophil CD64 for clinical infectious disease according to claim 1, characterized in that: in the S2 flow test, the method for processing the sample includes the following steps:
s2.1.1, respectively adding 20 μ L of CD64-PE monoclonal fluorescent antibody and 50 μ L of anticoagulated whole blood into the numbered test tubes, and mixing well;
s2.1.2 incubating at room temperature in dark for 15 min;
s2.1.3, adding 1mL of FACSLysin into the test tube, and uniformly mixing;
s2.1.4 incubating at room temperature in dark for 10 min;
s2.1.5, washing cells 2 times with 2mL of PBS buffer;
s2.1.6, cells were suspended in 450. mu.L PBS buffer.
4. The method of determining neutrophil CD64 for clinical infectious disease according to claim 1, characterized in that: in the S2 flow detection, the method for acquiring the fluorescence intensity of the lymphocytes, the monocytes and the neutrophils comprises the following steps:
s2.2.1, delineating lymphocytes, monocytes and neutrophils in an FSC/SSC scattergram, respectively;
s2.2.2, respectively establishing a histogram and displaying the geometric mean fluorescence intensity of the three groups of cells;
s2.2.3 10000 cells were taken per sample, and mean fluorescence intensity data for CD64 was obtained for each cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114441419A (en) * | 2022-01-29 | 2022-05-06 | 杭州翔宇医学检验实验室有限公司 | Stream type gate circling method and application |
| CN114624165A (en) * | 2022-03-10 | 2022-06-14 | 杭州翔宇医学检验实验室有限公司 | Method for measuring CD64 of neutrophils in blood sample |
| CN116042519A (en) * | 2023-03-31 | 2023-05-02 | 北京大学口腔医学院 | Classification, induction and activation method for repairing neutrophils and application thereof |
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| CN103604919A (en) * | 2013-11-05 | 2014-02-26 | 复旦大学附属华山医院 | Method for detecting activation peroid markers of T lymphocyte in human peripheral blood |
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2019
- 2019-11-28 CN CN201911186780.2A patent/CN110823789A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103604919A (en) * | 2013-11-05 | 2014-02-26 | 复旦大学附属华山医院 | Method for detecting activation peroid markers of T lymphocyte in human peripheral blood |
Non-Patent Citations (3)
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| 俸家富等: "《现代临床检验诊断大全》", 30 September 2013, 四川科学技术出版社 * |
| 许明峰: "脐带血中性粒细胞CD64指数在诊断胎膜新生儿感染的价值", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 闫佩毅等: "流式细胞术检测CD64指数及其在诊断细菌感染中的应用", 《检验医学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114441419A (en) * | 2022-01-29 | 2022-05-06 | 杭州翔宇医学检验实验室有限公司 | Stream type gate circling method and application |
| CN114441419B (en) * | 2022-01-29 | 2022-11-22 | 杭州翔宇医学检验实验室有限公司 | Flow type gate looping method and application |
| CN114624165A (en) * | 2022-03-10 | 2022-06-14 | 杭州翔宇医学检验实验室有限公司 | Method for measuring CD64 of neutrophils in blood sample |
| CN114624165B (en) * | 2022-03-10 | 2023-05-23 | 杭州翔宇医学检验实验室有限公司 | Method for measuring neutrophil CD64 in blood sample |
| CN116042519A (en) * | 2023-03-31 | 2023-05-02 | 北京大学口腔医学院 | Classification, induction and activation method for repairing neutrophils and application thereof |
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