CN112666062B - Method for combined detection of flow cytometry in humoral cell immunoassay - Google Patents
Method for combined detection of flow cytometry in humoral cell immunoassay Download PDFInfo
- Publication number
- CN112666062B CN112666062B CN202011308810.5A CN202011308810A CN112666062B CN 112666062 B CN112666062 B CN 112666062B CN 202011308810 A CN202011308810 A CN 202011308810A CN 112666062 B CN112666062 B CN 112666062B
- Authority
- CN
- China
- Prior art keywords
- cell
- body fluid
- specimen
- minutes
- flow cytometry
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000003018 immunoassay Methods 0.000 title claims abstract description 17
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 79
- 210000001124 body fluid Anatomy 0.000 claims abstract description 30
- 239000010839 body fluid Substances 0.000 claims abstract description 30
- 238000004458 analytical method Methods 0.000 claims abstract description 24
- 102000004127 Cytokines Human genes 0.000 claims abstract description 15
- 108090000695 Cytokines Proteins 0.000 claims abstract description 15
- 238000013394 immunophenotyping Methods 0.000 claims abstract description 10
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 239000003550 marker Substances 0.000 claims abstract description 7
- 239000000439 tumor marker Substances 0.000 claims abstract description 6
- 206010003445 Ascites Diseases 0.000 claims abstract description 5
- 206010048612 Hydrothorax Diseases 0.000 claims abstract description 5
- 206010036790 Productive cough Diseases 0.000 claims abstract description 5
- 239000012530 fluid Substances 0.000 claims abstract description 5
- 210000003802 sputum Anatomy 0.000 claims abstract description 5
- 208000024794 sputum Diseases 0.000 claims abstract description 5
- 210000002700 urine Anatomy 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 claims description 32
- 230000002159 abnormal effect Effects 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 17
- 239000013049 sediment Substances 0.000 claims description 15
- 238000010586 diagram Methods 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 11
- 230000001413 cellular effect Effects 0.000 claims description 9
- 210000000265 leukocyte Anatomy 0.000 claims description 9
- 206010028980 Neoplasm Diseases 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 7
- 210000003756 cervix mucus Anatomy 0.000 claims description 3
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000028709 inflammatory response Effects 0.000 claims 1
- 230000004044 response Effects 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 238000007689 inspection Methods 0.000 abstract description 5
- 206010061218 Inflammation Diseases 0.000 abstract description 2
- 201000011510 cancer Diseases 0.000 abstract description 2
- 238000003759 clinical diagnosis Methods 0.000 abstract description 2
- 239000007850 fluorescent dye Substances 0.000 abstract description 2
- 238000001215 fluorescent labelling Methods 0.000 abstract description 2
- 208000015181 infectious disease Diseases 0.000 abstract description 2
- 230000004054 inflammatory process Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 102000039446 nucleic acids Human genes 0.000 abstract description 2
- 108020004707 nucleic acids Proteins 0.000 abstract description 2
- 201000008827 tuberculosis Diseases 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract 1
- 239000004005 microsphere Substances 0.000 description 13
- 239000000126 substance Substances 0.000 description 10
- 239000011259 mixed solution Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 101000878581 Aplysia californica Feeding circuit activating peptides Proteins 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000012898 sample dilution Substances 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000757160 Homo sapiens Aminopeptidase N Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for combined detection in humoral cell immunoassay by flow cytometry, and relates to the technical field of immunoassay. The method for combined detection of the flow cytometry in the humoral cell immunoassay comprises the following steps: s1, collecting a body fluid specimen; s2, evaluating the quality and quantity of the specimens; s3, performing conventional cell population classification, multi-marker immunophenotyping, cytokine analysis and tumor marker examination; s4, analyzing results. The invention combines the technology of directly fluorescence labeling cell surface antibody and nucleic acid with conventional cell inspection and other methods to detect and analyze, can be used for detecting various diseases such as benign and malignant tumor, inflammation, bacterial and tuberculosis infection, has wide detection range of diseases and has higher clinical diagnosis and identification significance; the invention can adopt various body fluid detection, such as urine, sputum, alveolar lavage fluid, hydrothorax and ascites, dialysate, and the like, and has convenient and simple material acquisition and low cost.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a method for combined detection in humoral cell immunoassay by flow cytometry.
Background
The existing immunoassay technology adopts the steps that the position coordinates of each cell in a flow sample in a coordinate system taking different antigen molecular weights on the cell surface as coordinate axes are obtained; dividing cells in the flow sample into a plurality of cell populations according to the position coordinates; identifying a cell type of each of the plurality of cell populations; judging whether the position coordinates of the cells in each cell group are in a preset range corresponding to the cell type of the cell group or not; and determining the cell group as an abnormal group when the position coordinates of the cells in the cell group are not in the preset range corresponding to the cell type of the cell group as a judgment result. The DNA ploidy technique judges abnormal cell groups by acquiring different peaks from normal cells in a flow sample, and determining that the position coordinates are not in a preset range corresponding to the normal cell groups. The cytokine detection is carried out by capturing microsphere, combining with six specific antibodies on sample surface, combining with PE marked fluorescent antibody to form double antibody sandwich compound, detecting fluorescence intensity of compound, and obtaining content of six antibodies.
However, the prior art has the following disadvantages:
1. At present, blood analysis is lagged in the disease diagnosis process, and the three detection methods (immunophenotyping, DNA ploidy and cytokines) are earlier and more valuable than blood in the occurrence and development of diseases;
2. At present, three methods are independently detected, lack of comprehensive disease assessment means, cannot efficiently and accurately judge what diseases are, and have a small diagnosis range;
3. The traditional pathological diagnosis needs to take tissue biopsy by operation, has high cost and high risk, is great in damage to a patient and is painful, and can be analyzed by adopting a small amount of puncture samples by using fine needle puncture;
4. Detection of humoral cells under a common microscope: morphology is atypical, and cell numbers and quality are variable, abnormal cells are prone to missed detection or misjudgment of cell properties due to subjective awareness.
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides a method for combined detection in humoral cell immunoassay by flow cytometry, which solves the defects and shortcomings in the prior art.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme: a method of flow cytometry for joint detection in a humoral cellular immunoassay, the method comprising the steps of:
S1, collecting a body fluid specimen;
s2, evaluating the quality and quantity of the specimens;
S3, performing conventional cell population classification, multi-marker immunophenotyping, cytokine analysis and tumor marker examination;
S4, analyzing results.
Preferably, the body fluid sample includes, but is not limited to, urine, sputum, alveolar lavage fluid, hydrothorax and ascites, dialysate, vaginal secretion, and tissue mass puncture.
Preferably, the quality and quantity of the sample are evaluated as follows:
1) Observing the collection time, and inspecting the collected sample within four hours to obtain a fresh sample meeting the conditions;
2) Observing the collection amount of the specimen, wherein the specimen with the sample amount of more than 10ml is a specimen meeting the conditions;
3) And selecting a body fluid mode to operate by using the XN-2000 full-automatic modular blood body fluid analysis system of the Hizikian, and if the number of the white blood cells is more than 100x10 6/L, obtaining the sample meeting the number requirement.
Preferably, the conventional cell population classification is as follows:
1) Selecting a body fluid mode to be on-line by using a full-automatic modular blood body fluid analysis system XN-2000 of the Hissen health, and obtaining five categories of white blood cell number, red blood cell number and white blood cell;
2) After body fluid specimen 1300r/min was centrifuged for 5 minutes, the supernatant was removed, and after sediment smear was subjected to Rajie staining, classification counting was observed under a biological microscope.
Preferably, the multi-marker immunophenotyping is specifically as follows:
1) Centrifuging the body fluid specimen 1300r/min for 5 minutes, removing the supernatant, adding 2ml of PBS, uniformly mixing, centrifuging 1300r/min for 5 minutes, removing the supernatant, repeatedly washing for 3 times, and leaving sediment;
2) Taking a three-tube flow tube, wherein a test tube No. 1 comprises 50ul of specimen sediment, 320ul of fluorescent antibody FITC-CD, 16/56 20ul of PE-CD, 45 ul of PerCP-CD, 85 ul of PE-CY7-CD, 19 ul of APC-CD and 45 ul of APC-CY 7-CD; the test tube No. 2 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD7, 20ul of PE-CD117, 5ul of PE-CY7-CD19, 25 ul of APC-CD and 45 ul of APC-CY 7-CD; the test tube No. 3 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD45RA, 20ul of PE-CD34, 520ul of PerCP-CD, 7-CD13 ul of PE-CY, 5ul of APC-CD45RO, 5ul of APC-CY7-CD14 ul, mixing uniformly, keeping away from light for 15 minutes, adding 2ml of special membrane breaker, mixing uniformly, keeping away from light for 10 minutes, centrifuging 1300 to 5 minutes, discarding supernatant, adding 2ml of PBS, mixing uniformly, centrifuging 1300 to 5 minutes, discarding supernatant, adding 500ul of PBS into the test tube No. 1, adding 500ul of special dye into the test tube No. 2, mixing uniformly;
3) Analyzing by BDFACSCanto II flow cytometer BDFACSDiva Software software, setting different transverse and longitudinal coordinate channel marks to obtain a relation diagram between every two marks, and adjusting instrument voltage and compensation to enable the instrument to reach proper working conditions so as to obtain the number of cell groups shown by different marks and whether abnormal cells exist;
4) And (3) deriving experimental datse:Sub>A in BDFACSDivse:Sub>A Software software, importing experimental datse:Sub>A of experimental groups containing PI dye by using ModFitLT4.1 analysis software, reducing cell debris by all cells analyzed in se:Sub>A gate, setting an ordinate of SSC-A in se:Sub>A gate 2, setting an abscissse:Sub>A of APC-CY7-CD45, and repeatedly analyzing part of the circled cell population to observe whether abnormal cell population exists.
Preferably, the inverse gate technology is specifically as follows: the abnormal cell peak and in-frame abnormal cell population detected in the ModFit LT4.1 analysis software were selected in BDFACSDiva Software software on the same abscissa to select the same-position cell population P9.
Preferably, the cytokine assay is specifically as follows:
1) Centrifuging a body fluid specimen 1300r/min for 5 minutes, taking a supernatant, regulating voltage, adding 100ul of calibration microspheres (D) and 400ulPBS into a flow tube, and fully and uniformly mixing; performing analysis by using BD FACSCanto II flow cytometer upper machine BDFACSDiva Software software, setting FSC and SSC as Log in a parameter, and adjusting the voltages of the FSC and SSC to enable P1 to circle the beads group; adjusting the APC channel voltage to bring P2 to a mean value at APC of about 70000; checking the cytokines of the following 1:2 concentration calibration tube, observing whether the six groups are just on the rightmost side in the graph, and if the six groups are deviated, adjusting the FITC voltage to enable the six groups to be on the rightmost side;
2) The obtained voltage was used for the experimental voltage of the following calibration curve, which was set: calculating the number of required experimental human parts n=10 standard substances+1 negative control; opening a quantitative standard B, transferring the standard B into a centrifuge tube, and marking the tube as the highest concentration; 2ML sample dilution G resuspended standard, and left at room temperature for 15 min; mixing standard substances gently by using a suction head to avoid severe vibration, taking 10 experimental tubes with the highest concentration, respectively marking 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512, and adding 300 mu l of sample diluent G into each tube; 5. centrifuging the captured microsphere mixed solution A for 5 minutes by using a low-speed centrifuge at 200g, carefully sucking away the supernatant, sucking microsphere buffer solution H with the same volume as the sucked away supernatant, and incubating for 30 minutes in a dark place after vortex fully and uniformly mixing; adding 25 mu l of standard substance diluted in a gradient manner, 25 mu l of microsphere mixed solution and 25 mu l of fluorescent detection reagent into a standard substance tube, and incubating for 2.5 hours at room temperature in a dark place after vortex of all experimental tubes is fully mixed; 1ml of PBS solution is added into each tube of experiment tube, 200g is centrifuged for 5 minutes, the supernatant is carefully sucked away, 100 mu l of PBS solution is added into each tube, and the mixture is stood for waiting for inspection; fluorescent detection is carried out on the experimental tubes on the flow cytometer in a calibrated state according to the sequence of the standard substance, the negative control and the sample tube, and each experimental tube is required to be immediately detected after vortex mixing for 3-5 s; importing the result after detection into FCAP (FCAP) GUI (graphical user interface) exe software, and setting 2500pg/ml of the highest concentration;
3) Obtaining a calibration curve sample test: taking 50ul of capture microsphere mixed solution A, centrifuging for five minutes at 200g, sucking the supernatant, adding an equal volume of microsphere buffer solution H, mixing, standing for 30 minutes in a dark place, mixing, taking a flow tube, adding 25ul of mixed solution, 25ul of body fluid supernatant and 25ul of fluorescence detection reagent, mixing, adding 1ml of PBS solution in dark place for 2.5 hours, centrifuging for 5 minutes at 200g, sucking the supernatant, adding 100ul of PBS solution, mixing, analyzing by using BD FACSCanto II flow cytometer BD FACSDiva Software software, and introducing the obtained experimental data into FCAP. GUI. Exe software, and analyzing by using the calibration curve to obtain a result.
Preferably, the tumor marker examination is specifically as follows: after body fluid specimen 1300r/min is centrifuged for 5 minutes, the supernatant is taken and CEA, CA199 and CA125 are inspected by using a full-automatic electrochemiluminescence immunoassay analyzer C702.
Preferably, whether inflammatory or tumor reactivity is judged according to the result analysis and the report mode, and a model is built from multiple aspects of cell immunophenotyping, DNA ploidy, cytokines and tumor markers to provide reference basis for clinic.
(III) beneficial effects
The invention provides a method for combined detection in humoral cellular immune analysis by flow cytometry. The beneficial effects are as follows:
1. The invention combines the technology of directly fluorescence labeling cell surface antibody and nucleic acid with the methods of conventional cell inspection and the like to carry out joint detection analysis, can be used for detecting various diseases such as benign and malignant tumor, inflammation, bacterial infection, tuberculosis infection and the like, has a wide detection range of diseases, and has more standardization and higher clinical diagnosis and identification significance compared with the prior art that the cell property is judged by subjective consciousness error; the invention can adopt various body fluid detection, such as urine, sputum, alveolar lavage fluid, hydrothorax and ascites, dialysate, and the like, and has convenient and simple material acquisition and low cost.
2. The invention realizes the hole punching technology on the membrane so that the inside and outside of the membrane can be detected simultaneously, only the antibody mark and the sample are mixed, kept stand and centrifuged, the operation is simple, the time is saved, and the conventional detection can be realized.
3. According to the invention, a reverse gate technical scheme is established, so that the position of the abnormal cell group is more accurately found, the property of the abnormal cell group is clear, and the problem that a small amount of tumor cells are easy to miss in the traditional method is solved.
Drawings
FIG. 1 is a schematic diagram of an immunotyping No. 1 tube according to the present invention;
FIG. 2 is a schematic diagram of an immunotyping No. 2 tube according to the present invention;
FIG. 3 is a schematic diagram of a DNA ploidy experiment according to the present invention;
FIG. 4 is a schematic representation of a P9 abnormal cell population according to the present invention;
FIG. 5 is a schematic diagram showing the result of DNA ploidy false negative in the traditional method caused by the small number of tumor cells in the body fluid sample;
FIG. 6 is a schematic diagram showing the positive result of DNA ploidy heteroploidy with less application of the reverse gate technique in tumor cell count of the body fluid sample of the present invention;
FIG. 7 is a schematic diagram showing the adjustment of the APC channel voltage to 70000 according to the present invention;
FIG. 8 is a schematic diagram of 2500pg/ml modified PE channel voltage according to the present invention;
FIG. 9 is a graph of cytokine calibration according to the present invention;
FIG. 10 is a schematic diagram showing the results of cytokine detection according to the present invention;
Fig. 11 is a flow chart of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples:
As shown in fig. 1-11, an embodiment of the present invention provides a method for combined detection of flow cytometry in a humoral cellular immunoassay, the method comprising the steps of:
s1, collecting body fluid samples, wherein the body fluid samples comprise urine, sputum, alveolar lavage fluid, hydrothorax and ascites, dialysate, vaginal secretion and puncture tissue blocks;
S2, evaluating the quality and the quantity of the specimens, wherein the method specifically comprises the following steps of:
1) Observing the collection time, and inspecting the collected sample within four hours to obtain a fresh sample meeting the conditions;
2) Observing the collection amount of the specimen, wherein the specimen with the sample amount of more than 10ml is a specimen meeting the conditions;
3) Selecting a body fluid mode to be on-line by using a full-automatic module blood body fluid analysis system XN-2000 of the Hizimel, and if the number of the white blood cells is more than 100x10 6/L, obtaining a specimen meeting the number requirement;
S3, performing conventional cell population classification, multi-marker immunophenotyping, cytokine analysis and tumor marker examination;
conventional cell populations are classified as follows:
1) Selecting a body fluid mode to be on-line by using a full-automatic modular blood body fluid analysis system XN-2000 of the Hissen health, and obtaining five categories of white blood cell number, red blood cell number and white blood cell;
2) Centrifuging the body fluid specimen 1300r/min for 5 minutes, removing the supernatant, performing Rajie staining on the sediment smear, and observing and classifying and counting under a biological microscope;
the multi-marker immunophenotyping is specifically as follows:
1) Centrifuging the body fluid specimen 1300r/min for 5 minutes, removing the supernatant, adding 2ml of PBS, uniformly mixing, centrifuging 1300r/min for 5 minutes, removing the supernatant, repeatedly washing for 3 times, and leaving sediment;
2) Taking a three-tube flow tube, wherein a test tube No. 1 comprises 50ul of specimen sediment, 320ul of fluorescent antibody FITC-CD, 16/56 20ul of PE-CD, 45 ul of PerCP-CD, 85 ul of PE-CY7-CD, 19 ul of APC-CD and 45 ul of APC-CY 7-CD; the test tube No. 2 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD7, 20ul of PE-CD117, 5ul of PE-CY7-CD19, 25 ul of APC-CD and 45 ul of APC-CY 7-CD; the test tube No. 3 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD45RA, 20ul of PE-CD34, 20ul of PerCP-CD5, 5ul of PE-CY7-CD13, 5ul of APC-CD45RO, 5ul of APC-CY7-CD14, mixing uniformly, keeping away from light for 15 minutes, adding 2ml of special membrane breaker, mixing uniformly, keeping away from light for 10 minutes, centrifuging 1300 revolutions for 5 minutes, discarding supernatant, adding 2ml of PBS, mixing uniformly, centrifuging 1300 revolutions for 5 minutes, discarding supernatant, adding 500ul of PBS (main component propidium iodide) into the test tube No. 1 and 3, adding 500ul of special dye into the test tube No. 2, mixing uniformly;
3) Analyzing by BDFACSCanto II flow cytometer BDFACSDiva Software software, setting different abscissa channel marks to obtain a relation chart (CD 3 marked T lymphocyte, CD16/56 marked NK cell, CD45 marked lymphocyte, monocyte, granulocyte, CD8 marked inhibited/cytotoxic T cell, CD4 marked helper T cell, CD19 marked B lymphocyte, CD7 marked T lymphocyte, NK cell, CD117 marked marrow naive cell, CD2 marked T lymphocyte, NK cell, CD45RA marked non-sensitized T lymphocyte, CD45RO marked memory T lymphocyte, CD34 marked stem cell, progenitor cell, CD5 marked T lymphocyte, CD13 marked granulocyte, monocyte, CD14 marked monocyte), adjusting instrument voltage and compensation, so that the instrument reaches proper working conditions, thereby obtaining the number of cell groups shown by different marks and whether abnormal cells exist or not, as shown in figures 1 and 2;
4) Deriving experimental datse:Sub>A in BDFACSDivse:Sub>A Software software, importing experimental datse:Sub>A of an experimental group (namely se:Sub>A tube of FIG. 1 and se:Sub>A tube of FIG. 2) containing PI dye by using ModFitLT4.1 analysis software, reducing cell debris by all cells analyzed in se:Sub>A door loop of FIG. 1, setting an ordinate of SSC-A by se:Sub>A door of FIG. 2, and repeatedly analyzing part of cell groups in se:Sub>A circling manner to observe whether abnormal cell groups exist or not, wherein the result is shown in FIG. 3;
The reverse door setting technology comprises the following specific steps:
1) The method comprises the steps of realizing simultaneous marking inside and outside a membrane, analyzing a target cell group according to DNA marking in the membrane, reversely arranging a corresponding cell group on an analysis membrane, and marking once again according to the corresponding cell group to finish accurate DNA analysis;
2) The abnormal cell peak and the in-frame abnormal cell population detected in ModFit LT4.1 analysis software FIG. 3 are selected in BDFACSDiva Software software FIG. 1 to select the cell population P9 (FIG. 4) at the same position on the same abscissa and the same position on the same ordinate, and DNA analysis can obtain FIG. 5;
3) From the results, as shown in fig. 5 and 6, the negative results of a single peak detected by the traditional method are detected to be positive results of different ploidy of a plurality of peaks after being checked by the inverse gate method, so that whether abnormal cell groups exist or not can be more accurately judged, and the probability of missed detection under the condition of less tumor cell quantity is reduced;
cytokine analysis, specifically as follows:
1) Centrifuging a body fluid specimen 1300r/min for 5 minutes, taking a supernatant, regulating voltage, adding 100ul of calibration microspheres (D) and 400ulPBS into a flow tube, and fully and uniformly mixing; performing analysis by using BDFACSCanto II flow cytometer on-line BDFACSDiva Software software, setting FSC and SSC as Log in a parameter, and adjusting voltages of the FSC and SSC to enable P1 to encircle the beads group; right clicking the following double parameter diagram, and selecting a P1 gate by a show position; adjusting the APC channel voltage to bring P2 to a mean value at APC of about 70000 (FIG. 7); checking the cytokines of the following 1:2 concentration calibration tube, observing whether the hexa-cluster is just at the rightmost side in the graph, and if the hexa-cluster is deviated, adjusting the FITC voltage so that the hexa-cluster is at the rightmost side (as shown in FIG. 8);
2) The obtained voltage was used for the experimental voltage of the following calibration curve, which was set: calculating the number of required experimental human parts n=10 standard substances+1 negative control; opening a quantitative standard B, transferring the standard B into a centrifuge tube, and marking the tube as the highest concentration; 2ML sample dilution G resuspended standard, and left at room temperature for 15 min; mixing standard substances gently by using a suction head to avoid severe vibration, taking 10 experimental tubes with the highest concentration, respectively marking 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512, and adding 300 mu l of sample diluent G into each tube; 5. centrifuging the captured microsphere mixed solution A for 5 minutes by using a low-speed centrifuge at 200g, carefully sucking away the supernatant, sucking microsphere buffer solution H with the same volume as the sucked away supernatant, and incubating for 30 minutes in a dark place after vortex fully and uniformly mixing; adding 25 mu l of standard substance diluted in a gradient manner, 25 mu l of microsphere mixed solution and 25 mu l of fluorescent detection reagent into a standard substance tube, and incubating for 2.5 hours at room temperature in a dark place after vortex of all experimental tubes is fully mixed; 1ml of PBS solution is added into each tube of experiment tube, 200g is centrifuged for 5 minutes, the supernatant is carefully sucked away, 100 mu l of PBS solution is added into each tube, and the mixture is stood for waiting for inspection; fluorescent detection is carried out on the experimental tubes on the flow cytometer in a calibrated state according to the sequence of the standard substance, the negative control and the sample tube, and each experimental tube is required to be immediately detected after vortex mixing for 3-5 s; importing the result after detection into FCAP (FCAP) GUI (graphical user interface) exe software, and setting 2500pg/ml of the highest concentration;
3) A calibration curve (as in fig. 9) is obtained and the sample test: taking 50ul of capture microsphere mixed solution A, centrifuging 200g for five minutes, sucking away supernatant, adding an equal volume of microsphere buffer solution H, mixing, standing for 30 minutes in a dark place, mixing, taking a flow tube, adding 25ul of mixed solution, 25ul of body fluid supernatant and 25ul of fluorescence detection reagent, mixing, keeping away from the dark for 2.5 hours, adding 1ml of PBS solution, centrifuging 200g for 5 minutes, sucking away supernatant, adding 100ul of PBS solution, mixing, analyzing by using BD FACSCanto II flow cytometer BD FACSDiva Software software as shown in figure 10, introducing the obtained experimental data into FCAP. GUI. Exe software, and analyzing by using the calibration curve to obtain a result;
Tumor markers were examined as follows: centrifuging the body fluid specimen 1300r/min for 5 minutes, taking a supernatant, and carrying out CEA, CA199 and CA125 inspection by using a Germany Rogowski full-automatic electrochemiluminescence immunoassay instrument C702;
S4, the analysis of the results and the report form mode are shown in FIG 11, whether inflammatory reactivity or tumor reactivity is judged, and a model is built from multiple aspects of cell immunity typing, DNA ploidy, cytokines and tumor markers to provide reference basis for clinic.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for combined detection of flow cytometry in a humoral cellular immunoassay, characterized in that: the method comprises the following steps:
S1, collecting a body fluid specimen;
s2, evaluating the quality and quantity of the specimens;
S3, performing conventional cell population classification, multi-marker immunophenotyping, cytokine analysis and tumor marker examination;
s4, analyzing results;
The multi-marker immunophenotyping is specifically as follows:
Centrifuging the body fluid specimen 1300r/min for 5 minutes, removing the supernatant, adding 2ml of PBS, uniformly mixing, centrifuging 1300r/min for 5 minutes, removing the supernatant, repeatedly washing for 3 times, and leaving sediment;
Taking a three-tube flow tube, wherein a test tube No. 1 comprises 50ul of sample sediment, 20ul of fluorescent antibody FITC-CD3, 16/56 20ul of PE-CD, 45 ul of PerCP-CD, 7-CD 85 ul of PE-CY, 19 ul of APC-CD and 45 ul of APC-CY; the test tube No. 2 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD7, 20ul of PE-CD117, 5ul of PE-CY7-CD19, 25 ul of APC-CD and 45 ul of APC-CY 7-CD; the test tube No. 3 comprises 50ul of specimen sediment, 20ul of fluorescent antibody FITC-CD45RA, 20ul of PE-CD34, 20ul of PerCP-CD5, 20ul of PE-CY7-CD13, 5ul of APC-CD45RO, 5ul of APC-CY7-CD14, mixing uniformly, keeping away from light for 15 minutes, adding 2ml of special membrane breaker, mixing uniformly, keeping away from light for 10 minutes, centrifuging 1300 turns for 5 minutes, discarding supernatant, adding 2ml of PBS, mixing uniformly, centrifuging 1300 turns for 5 minutes, discarding supernatant, adding 500ul of PBS to test tubes No. 1 and No. 3, adding 500ul of special dye to test tube No. 2, mixing uniformly;
Analyzing by using BD FACSCanto II flow cytometer BD FACSDiva Software software, setting different abscissa channel marks to obtain a relation diagram between every two marks, adjusting instrument voltage and compensation to enable the instrument to reach proper working conditions, thereby obtaining the number of cell groups shown by different marks and whether abnormal cells exist or not;
deriving experimental datse:Sub>A in BD FACSDivse:Sub>A Software software, importing experimental datse:Sub>A of experimental groups containing PI dye by using ModFit LT 4.1 analysis software, reducing cell debris of all cells analyzed in se:Sub>A gate loop No. 1, setting an ordinate of SSC-A in se:Sub>A gate loop No. 2, setting an abscissse:Sub>A of APC-CY7-CD45, repeatedly analyzing part of cell groups, and observing whether abnormal cell groups exist or not;
the reverse gate technology is adopted, and the method concretely comprises the following steps:
1) The method comprises the steps of realizing simultaneous marking inside and outside a membrane, analyzing a target cell group according to DNA marking in the membrane, reversely arranging a corresponding cell group on an analysis membrane, and marking once again according to the corresponding cell group to finish accurate DNA analysis;
2) The abnormal cell peak and the in-frame abnormal cell population detected in the ModFit LT 4.1 analysis software were subjected to DNA analysis by selecting cell populations at the same positions on the same abscissa in BDFACSDiva Software software.
2. The method of flow cytometry in combination detection in a humoral cellular immunoassay according to claim 1, wherein: the body fluid specimen is urine, sputum, alveolar lavage fluid, hydrothorax and ascites, dialyzate, vaginal secretion and puncture tissue blocks.
3. The method of flow cytometry in combination detection in a humoral cellular immunoassay according to claim 1, wherein: the quality and quantity of the samples are evaluated as follows:
Observing the collection time, and inspecting the collected sample within four hours to obtain a fresh sample meeting the conditions;
Observing the collection amount of the specimen, wherein the specimen with the sample amount of more than 10ml is a specimen meeting the conditions;
3) And selecting a body fluid mode to operate by using the XN-2000 full-automatic modular blood body fluid analysis system of the Hizikian, and if the number of the white blood cells is more than 100x10 6/L, obtaining the sample meeting the number requirement.
4. The method of flow cytometry in combination detection in a humoral cellular immunoassay according to claim 1, wherein: the general cell population classification is specifically as follows:
selecting a body fluid mode to be on-line by using a full-automatic modular blood body fluid analysis system XN-2000 of the Hissen health, and obtaining five categories of white blood cell number, red blood cell number and white blood cell;
2) After body fluid specimen 1300r/min was centrifuged for 5 minutes, the supernatant was removed, and after sediment smear was subjected to Rajie staining, classification counting was observed under a biological microscope.
5. The method of flow cytometry in combination detection in a humoral cellular immunoassay according to claim 1, wherein: the tumor marker examination is specifically as follows: after body fluid specimen 1300r/min is centrifuged for 5 minutes, the supernatant is taken and CEA, CA199 and CA125 are inspected by using a full-automatic electrochemiluminescence immunoassay analyzer C702.
6. The method of flow cytometry in combination detection in a humoral cellular immunoassay according to claim 1, wherein: and judging whether the inflammatory response or the tumor response is carried out according to the result analysis and the report mode, and establishing a model from multiple aspects of cell immunophenotyping, DNA ploidy, cytokines and tumor markers to provide reference basis for clinic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011308810.5A CN112666062B (en) | 2020-11-20 | 2020-11-20 | Method for combined detection of flow cytometry in humoral cell immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011308810.5A CN112666062B (en) | 2020-11-20 | 2020-11-20 | Method for combined detection of flow cytometry in humoral cell immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112666062A CN112666062A (en) | 2021-04-16 |
| CN112666062B true CN112666062B (en) | 2024-05-24 |
Family
ID=75403443
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011308810.5A Active CN112666062B (en) | 2020-11-20 | 2020-11-20 | Method for combined detection of flow cytometry in humoral cell immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112666062B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113380318B (en) * | 2021-06-07 | 2023-04-07 | 天津金域医学检验实验室有限公司 | Artificial intelligence assisted flow cytometry 40CD immunophenotyping detection method and system |
| CN113419066B (en) * | 2021-08-23 | 2021-11-02 | 信纳克(北京)生化标志物检测医学研究有限责任公司 | Reagent composition for screening and/or diagnosing clonal diseases by one-step method and application thereof |
| CN113761456A (en) * | 2021-09-07 | 2021-12-07 | 杭州凯曼健康科技有限公司 | Immunofluorescence chromatography curve analysis method and device and electronic equipment |
| CN114136866A (en) * | 2021-10-28 | 2022-03-04 | 桂林优利特医疗电子有限公司 | Sperm nucleus DNA integrity detection method and detection kit |
| CN116794313B (en) * | 2023-08-18 | 2023-11-03 | 江西赛基生物技术有限公司 | Kit and method for simultaneously detecting three tumor markers based on flow cytometry |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275023A (en) * | 2019-05-13 | 2019-09-24 | 长春国科医工科技发展有限公司 | The method of joint-detection lung cancer tumor marker based on flow cytometry |
| CN110702589A (en) * | 2019-10-11 | 2020-01-17 | 南京吉鹏博生物科技有限公司 | Method for detecting exosome by flow cytometer |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007181462A (en) * | 2005-12-30 | 2007-07-19 | Centocor Inc | Method for measuring phenotype of cell |
-
2020
- 2020-11-20 CN CN202011308810.5A patent/CN112666062B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275023A (en) * | 2019-05-13 | 2019-09-24 | 长春国科医工科技发展有限公司 | The method of joint-detection lung cancer tumor marker based on flow cytometry |
| CN110702589A (en) * | 2019-10-11 | 2020-01-17 | 南京吉鹏博生物科技有限公司 | Method for detecting exosome by flow cytometer |
Non-Patent Citations (3)
| Title |
|---|
| XT-4000i全自动血细胞分析仪在体液细胞计数中的应用;王会平 等;《国际检验医学杂志》;第34卷(第15期);第2015-2016页 * |
| 流式细胞术在临床医学研究中的应用;崔巍 等;《临床输血与检验》;第7卷(第2期);第147-151页 * |
| 流式细胞术检测特发性血小板减少性紫癜患者血小板微粒;何敖林 等;《现代检验医学杂志》;第23卷(第1期);第74-76页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112666062A (en) | 2021-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112666062B (en) | Method for combined detection of flow cytometry in humoral cell immunoassay | |
| CA2674187C (en) | A high sensitivity multiparameter method for rare event analysis in a biological sample | |
| CN102246040B (en) | Method and device for diagnosing tuberculosis | |
| CN109266717B (en) | Method and device for detecting bacterial drug resistance through single cell analysis | |
| WO2008046292A1 (en) | Analysis method for 5-differential complete blood cell based on visual image | |
| CN115060882A (en) | Method for detecting rare cells by using non-rare cells | |
| Koken et al. | Determination of cut-off values for leucocytes and bacteria for urine flow cytometer (UF-100) in urinary tract infections | |
| CN105980852A (en) | Cell analysis method, system and apparatus | |
| US5252460A (en) | In vitro detection of ova, parasites, and other formed elements in stool | |
| CN115856302B (en) | Antibody composition for mature B cell tumor immunophenotyping and application thereof | |
| CN113484520A (en) | Molecular marker and application thereof in detection of angioimmunoblastic T-cell lymphoma | |
| Dimech et al. | Evaluation of an automated urinalysis system for testing urine chemistry, microscopy and culture | |
| JP7558248B2 (en) | Detection and reporting of neutrophil subpopulations | |
| Barr et al. | Routine flow cytometric diagnosis of lymphoproliferative disorders | |
| CN110726838A (en) | Circulating tumor cell bright field negative identification method based on immune microspheres | |
| CN117384289B (en) | Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit | |
| CN115389766A (en) | Marker for diagnosing whether neuroblastoma has bone marrow infiltration or not and application thereof | |
| CN118501451A (en) | Marker composition for evaluating T cell subpopulations and application thereof | |
| Mitchell et al. | The impact of technology on clinical pathology over the last 25 years | |
| Bayer et al. | Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies | |
| JP2004184103A (en) | Examination method for myelodysplastic syndrome | |
| CN118638231A (en) | Antibody composition and application thereof in detection of lymphocyte based on flow cytometry | |
| CN117092014A (en) | Method for rapidly identifying and correcting cold lectin interference | |
| Bungard et al. | Usefulness of the Sysmex XN 2000 Body-Fluid HF-BF Panel to detect early malignancy in pleural fluid and ascites fluid | |
| CN117288952A (en) | Antibody composition for detecting multiple myeloma based on full spectrum flow cytometry and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |