CN113959814A - Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis - Google Patents
Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis Download PDFInfo
- Publication number
- CN113959814A CN113959814A CN202111108369.0A CN202111108369A CN113959814A CN 113959814 A CN113959814 A CN 113959814A CN 202111108369 A CN202111108369 A CN 202111108369A CN 113959814 A CN113959814 A CN 113959814A
- Authority
- CN
- China
- Prior art keywords
- eversion
- detection
- platelets
- erythrocyte
- thrombus
- 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.)
- Pending
Links
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 66
- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 35
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 title claims abstract description 17
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 239000003147 molecular marker Substances 0.000 title description 5
- 210000004369 blood Anatomy 0.000 claims abstract description 15
- 239000008280 blood Substances 0.000 claims abstract description 15
- 210000001772 blood platelet Anatomy 0.000 claims description 56
- 238000002156 mixing Methods 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 claims description 8
- 238000000684 flow cytometry Methods 0.000 claims description 7
- 108010040476 FITC-annexin A5 Proteins 0.000 claims description 6
- 210000003462 vein Anatomy 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000010186 staining Methods 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000006907 apoptotic process Effects 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 3
- 108050008874 Annexin Proteins 0.000 claims 2
- 102000000412 Annexin Human genes 0.000 claims 2
- 238000003745 diagnosis Methods 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 7
- 238000013399 early diagnosis Methods 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 4
- 230000023555 blood coagulation Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 210000005259 peripheral blood Anatomy 0.000 abstract description 2
- 239000011886 peripheral blood Substances 0.000 abstract description 2
- 230000003331 prothrombotic effect Effects 0.000 abstract description 2
- 206010047249 Venous thrombosis Diseases 0.000 description 49
- 206010051055 Deep vein thrombosis Diseases 0.000 description 48
- 239000004793 Polystyrene Substances 0.000 description 36
- 229920002223 polystyrene Polymers 0.000 description 36
- 241001227561 Valgus Species 0.000 description 16
- 102100036523 Anoctamin-6 Human genes 0.000 description 10
- 101000928362 Homo sapiens Anoctamin-6 Proteins 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000003154 D dimer Substances 0.000 description 5
- 239000012148 binding buffer Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 210000003617 erythrocyte membrane Anatomy 0.000 description 5
- 108010052295 fibrin fragment D Proteins 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 4
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 208000010378 Pulmonary Embolism Diseases 0.000 description 3
- 230000010100 anticoagulation Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000002947 procoagulating effect Effects 0.000 description 3
- 102000035160 transmembrane proteins Human genes 0.000 description 3
- 108091005703 transmembrane proteins Proteins 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 238000002583 angiography Methods 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 230000007306 turnover Effects 0.000 description 2
- 108050008800 Anoctamin-6 Proteins 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 201000000552 Scott syndrome Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 229940030606 diuretics Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000005980 lung dysfunction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009057 passive transport Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 150000008106 phosphatidylserines Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000012488 skeletal system development Effects 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 201000002282 venous insufficiency Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 208000002670 vitamin B12 deficiency Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N15/1404—Handling flow, e.g. hydrodynamic focusing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
- G01N15/1431—Signal processing the electronics being integrated with the analyser, e.g. hand-held devices for on-site investigation
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Dispersion Chemistry (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Signal Processing (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses application of erythrocyte and platelet phosphatidylserine eversion as molecular markers in detecting thrombosis. The invention provides a new index for diagnosing thrombus by taking the eversion of erythrocyte and platelet Phosphatidylserine (PS) as molecular markers of thrombus formation on the basis of a new theory that the eversion increase of erythrocyte and platelet PS is an important mechanism for the development of thrombus. The detection sample is peripheral blood, the material is easy to obtain, 2 microliters of whole blood is needed for each detection, the detection time is 10 minutes, and the required instrument is a flow cytometer. The method has the advantages of small sample consumption, simple and convenient detection, rapidness, sensitivity and low cost, is suitable for being developed in clinical laboratory, provides a new detection technology for early diagnosis of thrombotic diseases, and provides a more reliable diagnosis basis for diagnosis of prothrombotic state by combining with clinical common blood coagulation detection indexes.
Description
Technical Field
The invention relates to a molecular marker for detecting thrombosis, in particular to application of erythrocyte and platelet phosphatidylserine eversion as the molecular marker in detecting thrombosis. The invention belongs to the technical field of medicines.
Background
Deep Venous Thrombosis (DVT) is a venous reflux disorder caused by abnormal coagulation of blood in Deep veins, is the third leading cause of cardiovascular-related disease death following myocardial infarction and stroke, and has an increasing incidence. The susceptibility to complicated fatal Pulmonary Embolism (PE), premature pulmonary dysfunction and the like have seriously affected the life health and the quality of life of patients. At present, although comprehensive treatment methods such as thrombolysis, anticoagulation, coagulation removal and blood vessel expansion and the like and treatment methods such as placing a vena cava filter and the like are continuously innovated, the treatment of the diseases is greatly progressed, however, the thrombosis symptoms of DVT patients are hidden, and the clinical early warning index of thrombosis is lacked.
Currently, the main diagnostic aids for DVT are angiography, ultrasound examination and D-Dimer (D-Dimer) detection, which can only diagnose thrombus that has already occurred with a relatively delayed diagnosis time. Angiography, although the "gold standard" for DVT diagnosis, is relatively expensive to examine and has invasive lesions. D-dimers are specific degradation products of cross-linked fibrin monomers and are indicative of secondary fibrinolysis. Is a DVT diagnostic index widely used clinically at present, has high sensitivity, has good clinical application value in the aspects of diagnosis of DVT, PE and Disseminated Intravascular Coagulation (DIC) and thrombolytic therapy detection, and has generally accepted clinical diagnostic value as a biological index of thrombosis. The D-dimer has important significance for diagnosis, curative effect evaluation and prognosis judgment of thrombotic diseases as a main biological index for diagnosing DVT, but has many defects in the aspects related to methodology, quality control, standardization and clinical application. (1) Insufficient specificity: d-dimer is not a specific index of deep venous thrombosis, and the expression of D-dimer can be increased in diseases such as infection, myocardial infarction, trauma and tumor. (2) Insufficient early diagnosis ability: the rise in D-dimer suggests that the organism has formed a thrombus and that the diagnosis time is relatively late. (3) Incompatibilities of detection results of different reagents: the monoclonal antibodies prepared aiming at different fragments have different binding capacities on different fragments in the same plasma, so that at present, no monoclonal antibody with international unified standard exists, no unified reference method exists, and the immunoturbidimetry method adopted on an automatic hemagglutination instrument is influenced by the characteristics of latex particles, so that the specificity difference of the monoclonal antibodies and the reagent production process are different, and the detection results of all reagents are lack of comparability.
Therefore, aiming at the defects of the prior art, the exploration of early diagnosis and monitoring indexes has important significance for the prevention and treatment of the diseases.
Disclosure of Invention
The invention aims to provide a molecular marker for early diagnosis of thrombus, in particular deep vein thrombus.
In order to achieve the purpose, the invention adopts the following technical means:
firstly, the invention provides the application of the eversion of erythrocyte and platelet phosphatidylserine as molecular markers of thrombus formation in the preparation of reagents or devices for detecting the thrombus formation.
Secondly, the invention also provides the application of the instrument for detecting the eversion of the red blood cells and the platelet phosphatidylserine in the preparation of a device for detecting the formation of thrombus.
Wherein, the instrument for detecting the eversion of the red blood cells and the platelet phosphatidylserine is preferably a flow cytometer.
Preferably, the device further comprises a fluorescent-labeled Annexin V.
Wherein, the fluorescence labeled Annexin V is Annexin V-FITC preferably.
Preferably, the thrombus is a deep vein thrombus.
Wherein, preferably, the device is used for detecting the condition of the eversion of the phosphatidylserine in the red blood cells and the platelets and comprises the following steps:
1) dyeing:
(1) preparing 1.5ml of EP tubes, and respectively marking the tubes as dyed tubes and undyed tubes, wherein the undyed tubes are used as the setting of a negative gate in flow detection analysis;
(2) adding 50 mu L of binding solution in the apoptosis kit into an EP tube marked with staining, then adding 5 mu L of Annexin V-FITC for uniformly mixing, then adding 2 mu L of fully and uniformly mixed whole blood for uniformly mixing, and incubating for 10 minutes at room temperature in a dark place; adding 2 mu L of whole blood into 50 mu L binding buffer of an unstained tube system;
(3) after 10 minutes, 350 mu L PBS is added into each tube, and the tubes are transferred to a flow tube and then are arranged on a machine; 2) flow cytometry analysis
(1) Starting up: starting a Cytek flow cytometer, performing Prime for 3 times, starting FJCE software, and opening a FlowJo CE window;
(2) setting parameters: clicking a green acquisition key to open a data display window of the flow cytometer, setting a collection mode to be a logarithmic mode, setting an FSC threshold value to be 0.7, and stopping collection after 10000 events are collected;
(3) loading: collecting samples at LOW speed of RUN, LOW (12 μ L/min), adjusting voltage of FSC, SSC and BluFL1 channels to proper position to divide platelets and red blood cells into 2 groups;
(4) collecting cells: starting collection by pressing a collection key, respectively gating red blood cells and platelets in FlowJo after collection, setting an unstained negative gate to be 0.1%, and comparing with a staining tube;
(5) obtaining PS eversion percentages of erythrocytes and platelets;
after saving the data, the instrument was cleaned and shut down.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention is based on a new theory that the increase of the PS valgus of the erythrocytes and the platelets is an important mechanism for the development of the thrombus, provides the PS valgus of the erythrocytes and the platelets as molecular markers of the thrombus formation, and is a new index for diagnosing the thrombus.
2. Annexin V is used as a small molecular probe for detection, and the eversion proportion of erythrocytes and platelets PS is detected by a flow cytometer, so that the detection method is more sensitive and specific as early diagnosis of thrombosis.
3. The detection sample is peripheral blood, the material is easy to obtain, 2 microliters of whole blood is needed for each detection, the detection time is 10 minutes, and the required instrument is a flow cytometer. The method has the advantages of small sample consumption, simple and convenient detection, rapidness, sensitivity and low cost, is suitable for being developed in clinical laboratory, provides a new detection technology for early diagnosis of thrombotic diseases, and provides a more reliable diagnosis basis for diagnosis of prothrombotic state by combining with clinical common blood coagulation detection indexes.
Drawings
FIG. 1 is a flow cytometer for analyzing the PS valgus condition of red blood cells and platelets;
wherein, A: the flow cytometry shows a scatter diagram of red blood cells and platelets, the upper right cell mass is red blood cells, and the lower left cell mass is platelets; b: histograms of red blood cells from DVT patients and healthy controls, the black line indicated by the "arrow" represents red blood cells from DVT patients, and the remaining black line represents red blood cells from healthy controls, as shown in the figure, DVT patients have higher PS valgus levels than red blood cells from healthy controls; c: histograms of platelets from DVT patients and healthy controls, the black line indicated by the "arrow" representing platelets from DVT patients, the remaining black line representing platelets from healthy controls, as shown by the higher PS valgus level of DVT patients compared to platelets from healthy controls; percentage of PS valgus of erythrocytes in DVT patients and healthy controls; percent PS eversion of platelets in DVT patients and healthy controls; f: the ratio of the PS valgus level of erythrocytes in DVT patients and in healthy control groups to the ratio of the PS valgus level of platelets in DVT patients and in healthy control groups. Results are shown by mean ± standard deviation.
FIG. 2 is a graph of laser confocal analysis of PS valgus levels of red blood cells and platelets;
wherein, A: red blood cells of a fluorescently labeled healthy control group; b: fluorescently labeled red blood cells of a DVT patient; c: platelets of a fluorescently labeled healthy control group; d: fluorescently labeled platelets of a DVT patient;
FIG. 3 is a time measurement of red blood cell and platelet recalcification in DVT patients and healthy controls;
wherein, A: the red blood cell recalcification time of the DVT patient is shortened compared with that of a healthy control group; b: the platelet recalcification time of the DVT patient is shortened compared with that of a healthy control group; results are shown as mean ± standard deviation;
FIG. 4 shows that Western Blotting detects the expression level of TMEM16F protein which can regulate PS eversion on erythrocyte membranes of normal human and deep venous thrombosis patients;
wherein, A: western Blotting results; b: is a bar graph of the results of graph A.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 Experimental study of erythrocyte and platelet Phosphatidylserine (PS) eversion as a marker for thrombosis
Research content and method
1. Study subjects: 50 healthy volunteers were recruited in cooperation with the affiliated hospital clinical laboratory and clinical department. Establishing a reference interval of the normal population erythrocyte and platelet PS eversion proportion. 50 patients with venous thrombosis were recruited.
The DVT suppository patients are first-diagnosed patients in fifth hospital affiliated to Harbin medical university in 9 months to 9 months in 2019, and the average age of the DVT suppository patients is 65.45 +/-10.64 years in 50 cases, 27 cases and 23 cases of women in the DVT patients. The healthy control group had 50 cases, 25 men and 25 women, and the mean age was 65.8 ± 7.19 years. Inclusion subject exclusion criteria: no cardiovascular disease, no history of drug use before detection, no risk factors of cardiovascular disease such as diabetes and hypertension; no malignant tumor and systemic diseases, no pregnancy, no ferrum, folic acid, vitamin B12 deficiency, no history of blood transfusion within six months, no acute and chronic infection, and no drug for affecting hemostatic function; the patient did not receive diuretics, hormones and other immunosuppressive agents.
2. Flow cytometry for detecting PS (Poly styrene) eversion on surfaces of red blood cells and platelets
2.1 blood collection and anticoagulation, namely collecting 2mL of blood by using an anticoagulation tube containing 3.8 percent of sodium citrate for vein blood collection, and fully and uniformly mixing the anticoagulation agent and the blood.
2.2 dyeing:
(1) preparing 1.5ml of EP tubes, and respectively marking the tubes as dyed tubes and undyed tubes, wherein the undyed tubes are used as the setting of a negative gate in flow detection analysis;
(2) adding 50 mu L of binding buffer in an apoptosis kit into an EP tube marked with staining, then adding 5 mu L of Annexin V-FITC for uniformly mixing, then adding 2 mu L of fully and uniformly mixed whole blood, uniformly mixing, and incubating for 10 minutes at room temperature in a dark place; the unstained tube was added to 50. mu.L binding buffer with 2. mu.L whole blood.
(3) After 10 minutes 350. mu.L PBS was added to each tube and transferred to the flow tube and then loaded onto the machine.
2.3 flow cytometry analysis
(1) Starting up: starting a Cytek flow cytometer, performing Prime for 3 times, starting FJCE software, and opening a FlowJo CE window;
(2) setting parameters: clicking a green acquisition key to open a data display window of the flow cytometer, setting a collection mode to be a logarithmic mode, setting an FSC threshold value to be 0.7, and stopping collection after 10000 events are collected;
(3) loading: samples were collected at LOW RUN, LOW (12 μ L/min) rates, and platelets and red blood cells were grouped into 2 groups by adjusting the voltage levels of the FSC, SSC and BluFL1 channels (FSC: 0.1X 5.8; SSC: 320; BluFL 1: 480);
(4) collecting cells: pressing the collect key starts collecting. After collection, red blood cells and platelets were gated in FlowJo, and the unstained negative gate was set to 0.1% and compared to the stained tube;
(5) obtaining PS eversion percentages of erythrocytes and platelets;
(6) after saving the data, the instrument was cleaned and shut down.
3. Laser confocal microscope detection of PS (Poly styrene) eversion on surfaces of red blood cells and platelets
(1) A1000 XRBC suspension was prepared by adding 198. mu.L Tyrode buffer to the tube and 2. mu.L packed red blood cells. Taking a test tube, adding 90 mu L of Tyrode buffer, adding 10 mu L of the 100 XRBC suspension, centrifuging for 1 minute at 600r, removing supernatant, and adding 25 mu L of binding buffer for resuspension;
(2) dyeing: adding 50 mu L binding buffer into the test tube, adding 50 mu L Annexin V-FITC, mixing uniformly, adding the prepared erythrocyte suspension, mixing uniformly, keeping out of the sun, and incubating for 15 minutes at room temperature;
(3) washing erythrocytes once, discarding supernatant, adding 100 μ L Tyrode buffer, mixing well, plating, keeping out of the sun, and loading on a machine;
(4) and (4) observing by a confocal microscope.
(II) results of the experiment
1. Detection of PS valgus of groups of red blood cells and platelets by flow cytometry
The flow cytometry detects the PS eversion percentage of erythrocyte membranes of patients with deep venous thrombosis and normal people. The PS valgus of the erythrocytes of the DVT patients is 9.4 +/-0.9 percent and is obviously higher than that of a healthy control group by 0.75 +/-0.2 percent (P is less than 0.001); the platelets in DVT patients have PS (platelet-dependent) eversion level of 24.1 +/-3.0%, which is obviously higher than that of healthy volunteers by 6.3 +/-0.3%, (P <0.001), and the fold of PS eversion of erythrocytes in DVT patients is obviously higher than that of platelets, as shown in Table 1 and figure 1.
TABLE 1 Primary data for PS eversion of the surface of erythrocytes in DVT patients and healthy persons
2. Laser confocal microscope for observing PS (Poly styrene) valgus condition of red blood cells and platelets
To further verify the PS eversion of erythrocytes and platelets, we observed with a confocal laser microscope. Taking red blood cells of normal human and deep venous thrombosis patients, the PS valgus increase (green) of the red blood cells of the DVT patients can be seen in figure 2B, and the red blood cells of the normal control group are negative. Platelets from normal human and deep venous thrombosis patients were incubated with Annexin V-FITC and observed to show significant increase in PS valgus (green) in DVT patients, as shown in FIG. 2.
Comparison of erythrocyte and platelet procoagulant Activity in DVT patients with healthy humans
To demonstrate that the procoagulant activity of erythrocytes and platelets in DVT patients is dependent on PS valgus, peripheral venous blood from healthy control groups and DVT patients was collected and their erythrocytes and platelets were isolated in this experiment, and an erythrocyte suspension was prepared (10)9Individual cells/mL) and platelet suspension (10)8Individual cells/mL), then adding platelet-free plasma prepared in advance and calcium ions, recording the clotting time with a coagulometer, and measuring the erythrocyte and platelet procoagulant activities of DVT patients and healthy controls, the results being expressed in seconds. The red blood cell recalcification time of the DVT patient is 257.9 +/-45.2 seconds, which is obviously shortened compared with 354.9 +/-47.2 seconds of a healthy control group (P)<0.001); the recalcification time of the platelets of the DVT patient is 261.0 +/-60.3 seconds, which is obviously shortened compared with 348.7 +/-57.9 seconds of a healthy control group (P)<0.001), see fig. 3.
Second, research on PS valgus mechanism of red blood cells and platelets of DVT patients
We have conducted preliminary studies on transmembrane proteins regulating erythrocytes, in order to complete the phosphatidylserine evagination mechanism of erythrocytes. Transmembrane protein16F (TMEM 16F ) also called Anocamin 6(ANO6) is a protein composed of Ca2+An activated phospholipid turnover promoter enzyme belonging to the family of transmembrane protein Anocamin/TMEM 16. It has dual activities, both phospholipid turnover and ion channel activities. TMEM16F allows passive transport of phospholipids along its chemical gradient, transferring phospholipids bi-directionally between the leaflets of the plasma membrane, thereby flipping negatively charged PS from the intimal leaflet to the adventitial leaflet, and thus it is Ca on the surface of many eukaryotic cells2+Transmembrane proteins necessary for the eversion of activated phosphatidylserine. TMEM16F is widely expressed in a variety of cells (e.g., platelets, bone cells and immune cells) and mediates physiological processes such as blood clotting, skeletal development and viral infection. And when TMEM16F is mutated it will lead to the hemorrhagic disease Scott syndrome. We therefore explored whether TMEM16F would mediate PS eversion of erythrocytes and platelets in DVT patients. The expression level of TMEM16F protein which can regulate PS eversion on erythrocyte membranes of normal human and deep venous thrombosis patients is compared by using a Western Blotting technology, and the result shows that the expression level of the TMEM16F protein on the erythrocyte membranes of the DVT patients is higher than that of a healthy control group, and P is higher than that of the TMEM16F protein on the erythrocyte membranes of the healthy human and deep venous thrombosis patients<0.05, see fig. 4.
Claims (8)
1. The use of erythrocyte and platelet phosphatidylserine eversion as molecular markers of thrombosis in the preparation of reagents or devices for detecting thrombosis.
2. The use according to claim 1, wherein the thrombus is a deep vein thrombus.
3. Use of an apparatus for the detection of the eversion of phosphatidylserine in erythrocytes and platelets for the preparation of a device for the detection of thrombosis.
4. The use according to claim 2, wherein the apparatus for detecting the eversion of red blood cells and platelet phosphatidylserine is a flow cytometer.
5. The use of claim 2, wherein said device further comprises fluorescently labeled annexin v.
6. The use according to claim 2, wherein the fluorescently labeled annexin v is annexin v-FITC.
7. The use according to claim 3, wherein the thrombus is a deep vein thrombus.
8. Use according to any one of claims 3 to 7, wherein the device is for the detection of the condition of erythrocyte and platelet phosphatidylserine eversion, comprising the steps of:
1) dyeing:
(1) preparing 1.5ml of EP tubes, and respectively marking the tubes as dyed tubes and undyed tubes, wherein the undyed tubes are used as the setting of a negative gate in flow detection analysis;
(2) adding 50 mu L of binding solution in the apoptosis kit into an EP tube marked with staining, then adding 5 mu LannexinV-FITC for uniformly mixing, then adding 2 mu L of fully and uniformly mixed whole blood, uniformly mixing, and incubating for 10 minutes at room temperature in a dark place; adding 2 mu L of whole blood into the unstained tube system of 50 mu Lbrindingbuffer;
(3) after 10 minutes, adding 350 mu LPBS into each tube, transferring to a flow tube, and then loading on a machine;
2) flow cytometry analysis
(1) Starting up: starting a Cytek flow cytometer, performing Prime for 3 times, starting FJCE software, and opening a FlowJoCE window;
(2) setting parameters: clicking a green acquisition key to open a data display window of the flow cytometer, setting a collection mode to be a logarithmic mode, setting an FSC threshold value to be 0.7, and stopping collection after 10000 events are collected;
(3) loading: collecting samples at LOW speed of RUN, LOW (12 μ L/min), adjusting voltage of FSC, SSC and BluFL1 channels to proper position to divide platelets and red blood cells into 2 groups;
(4) collecting cells: starting collection by pressing a collection key, respectively gating red blood cells and platelets in FlowJo after collection, setting an unstained negative gate to be 0.1%, and comparing with a staining tube;
(5) obtaining PS eversion percentages of erythrocytes and platelets;
(6) after saving the data, the instrument was cleaned and shut down.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111108369.0A CN113959814A (en) | 2021-09-22 | 2021-09-22 | Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111108369.0A CN113959814A (en) | 2021-09-22 | 2021-09-22 | Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113959814A true CN113959814A (en) | 2022-01-21 |
Family
ID=79461905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111108369.0A Pending CN113959814A (en) | 2021-09-22 | 2021-09-22 | Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113959814A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992015886A1 (en) * | 1991-03-08 | 1992-09-17 | Board Of Regents, The University Of Texas System | Detection of early platelet activation and prediagnosis of thrombotic events |
| US20060199239A1 (en) * | 2005-03-01 | 2006-09-07 | Gurbel Paul A | Assessment of cardiac health and thrombotic risk in a patient |
| CN107860702A (en) * | 2017-11-09 | 2018-03-30 | 傅应云 | A kind of method for detecting pulmonary embolism |
| CN107952073A (en) * | 2016-10-17 | 2018-04-24 | 中国科学院动物研究所 | A kind of medicine for adjusting biologically active pdgf, screening technique and application thereof |
| CN108159421A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | Application of phosphatidylserine blocking agent in preparation of medicine for treating diseases related to platelet quantity reduction |
-
2021
- 2021-09-22 CN CN202111108369.0A patent/CN113959814A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992015886A1 (en) * | 1991-03-08 | 1992-09-17 | Board Of Regents, The University Of Texas System | Detection of early platelet activation and prediagnosis of thrombotic events |
| US20060199239A1 (en) * | 2005-03-01 | 2006-09-07 | Gurbel Paul A | Assessment of cardiac health and thrombotic risk in a patient |
| CN107952073A (en) * | 2016-10-17 | 2018-04-24 | 中国科学院动物研究所 | A kind of medicine for adjusting biologically active pdgf, screening technique and application thereof |
| CN107860702A (en) * | 2017-11-09 | 2018-03-30 | 傅应云 | A kind of method for detecting pulmonary embolism |
| CN108159421A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | Application of phosphatidylserine blocking agent in preparation of medicine for treating diseases related to platelet quantity reduction |
Non-Patent Citations (2)
| Title |
|---|
| 王秦等: "磷脂酰丝氨酸及其结合配体与尿毒症血液高凝状态的相关性", 《中华临床医师杂志》, 15 August 2012 (2012-08-15), pages 1 - 3 * |
| 赵玲等: "血小板参数和血小板聚集功能检测对2型糖尿病治疗后检测的临床价值探讨", 《国际检验医学杂志》, 31 May 2021 (2021-05-31), pages 1164 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109596843B (en) | A kind of assay kit of serum amyloid A protein | |
| CN108956998A (en) | A kind of heparin-binding protein assay kit and measuring method using immunofluorescence dry type quantitative method | |
| CN110726846B (en) | Application of HBP protein as diagnostic marker of Kawasaki disease | |
| Manivannan et al. | Diagnosis of paroxysmal nocturnal hemoglobinuria: recent advances | |
| US20080213800A1 (en) | Method for Examing Interstitital Cystitis | |
| CN115561468B (en) | Method for assessing risk of suffering from tumor or specific tumor | |
| CN113959814A (en) | Application of erythrocyte and platelet phosphatidylserine eversion as molecular marker in detection of thrombosis | |
| CN114636826B (en) | Application of CD177+ neutrophils in preparation of detection product for neonatal necrotizing enterocolitis | |
| Mowafy et al. | Role of platelet indices and antiplatelet antibody in differentiating immune thrombocytopenic purpura from other causes of thrombocytopenia | |
| CN104198694A (en) | Diagnostic kit and method for identifying tuberculosis and tumors by using same | |
| JP2000193662A (en) | Measuring reagent for cystatin c in urine, diagnostic method and kit | |
| CN101109752B (en) | Method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method | |
| JP2553606B2 (en) | Method for separating and using density-specific blood cells | |
| CN109490556A (en) | AGP1, ORM2 and C9 are distinguishing the application in tuberculous pleural effusion and malignant pleural effusion | |
| Galante et al. | Infection with Helicobacter pylori and leukocyte response in patients with myocardial infarction | |
| Heffner et al. | Role of the Peripheral Intravenous Catheter in False‐positive D‐dimer Testing | |
| CN115389766B (en) | Marker for diagnosing whether neuroblastoma is subjected to bone marrow infiltration and application thereof | |
| US20230366880A1 (en) | Methods and kits for diagnosing covid-19 disease | |
| JP2005221323A (en) | Method for detecting osteomyelodysplasia syndrome | |
| Al-fituri et al. | Evaluation of using urine strip in diagnosis of children's meningitis in Benghazi-Libya | |
| CN110361531B (en) | Experimental method for detecting particle coagulation promoting activity | |
| WO2024098369A1 (en) | Parkinson's disease in-vitro diagnostic kit based on dna hexahedron and use thereof | |
| Drochnerytė et al. | COMPARATIVE ANALYSIS OF STANDARDISED AND NON-STANDARDISED MICROSCOPY METHODS OF URINE SEDIMENTS | |
| Abe et al. | Measurement of fibrinogen degradation products (FDP) in serum of normal and tumor-bearing rats | |
| CN116338191A (en) | Lung cancer exosome protein marker based on flow technology and application thereof in aspect of lung cancer screening |
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 |