CN117737193A - Thrombus elastography heparinase cup, and preparation method and application thereof - Google Patents
Thrombus elastography heparinase cup, and preparation method and application thereof Download PDFInfo
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- CN117737193A CN117737193A CN202311682340.2A CN202311682340A CN117737193A CN 117737193 A CN117737193 A CN 117737193A CN 202311682340 A CN202311682340 A CN 202311682340A CN 117737193 A CN117737193 A CN 117737193A
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- 108010022901 Heparin Lyase Proteins 0.000 title claims abstract description 57
- 238000002091 elastography Methods 0.000 title claims description 13
- 238000002360 preparation method Methods 0.000 title claims description 12
- 208000007536 Thrombosis Diseases 0.000 title abstract description 19
- 238000010227 cup method (microbiological evaluation) Methods 0.000 title description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 72
- 238000001514 detection method Methods 0.000 claims abstract description 48
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 19
- 239000012190 activator Substances 0.000 claims abstract description 18
- 239000001110 calcium chloride Substances 0.000 claims abstract description 18
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 18
- 102100024025 Heparanase Human genes 0.000 claims abstract description 15
- 108010037536 heparanase Proteins 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000002699 waste material Substances 0.000 claims abstract description 8
- 230000003213 activating effect Effects 0.000 claims abstract description 4
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- 239000005995 Aluminium silicate Substances 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 5
- 229920002307 Dextran Polymers 0.000 claims description 5
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 5
- 235000012211 aluminium silicate Nutrition 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 claims description 5
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 claims description 4
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 claims description 4
- 229920002079 Ellagic acid Polymers 0.000 claims description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 4
- 229910052782 aluminium Inorganic materials 0.000 claims description 4
- 229960002852 ellagic acid Drugs 0.000 claims description 4
- 235000004132 ellagic acid Nutrition 0.000 claims description 4
- 239000011888 foil Substances 0.000 claims description 4
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 claims description 4
- 239000003223 protective agent Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 230000002792 vascular Effects 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 239000005909 Kieselgur Substances 0.000 claims 1
- 239000005030 aluminium foil Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 16
- 239000008280 blood Substances 0.000 abstract description 16
- 238000003860 storage Methods 0.000 abstract description 7
- 239000001509 sodium citrate Substances 0.000 abstract description 5
- 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 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 21
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 17
- 229960002897 heparin Drugs 0.000 description 17
- 229920000669 heparin Polymers 0.000 description 17
- 230000035602 clotting Effects 0.000 description 13
- 238000003908 quality control method Methods 0.000 description 12
- 235000011148 calcium chloride Nutrition 0.000 description 11
- 206010053567 Coagulopathies Diseases 0.000 description 10
- 230000023555 blood coagulation Effects 0.000 description 10
- 239000004019 antithrombin Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 4
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 4
- 239000003114 blood coagulation factor Substances 0.000 description 4
- 230000020764 fibrinolysis Effects 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
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- 238000005345 coagulation Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000003466 welding Methods 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 101000712605 Theromyzon tessulatum Theromin Proteins 0.000 description 1
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- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a heparinase cup with a thrombus elasticity map, which is characterized by comprising a reagent card, wherein the reagent card comprises a sample bin, a microfluidic channel, a quantifying chamber, an activator channel, a detection channel and a waste liquid bin, the sample bin is communicated with the microfluidic channel, the microfluidic channel is communicated with the quantifying chamber, and the quantifying chamber is communicated with the waste liquid bin; the quantifying chamber is communicated with the activator channel and the detection channel, and the microfluidic channel is communicated with the three channels; the three channels are respectively provided with freeze-dried balls coated with an activating agent and freeze-dried balls coated with calcium chloride; comprises a heparanase coated freeze-dried ball and a calcium chloride coated freeze-dried ball; and an empty channel. The freeze-dried balls provided by the invention effectively solve the problems of cold chain transportation, low-temperature preservation, short storage period and the like. The fully-closed matched instrument is adopted to complete detection, and only the collected sodium citrate anticoagulated venous whole blood collection tube is inserted into the reagent card during the detection, so that the reagent card can be directly operated, the operation is simple and convenient, and the detection is rapid.
Description
Technical Field
The invention relates to the technical field of medical appliances, in particular to a heparinase cup with a thrombus elasticity chart, a preparation method and application thereof.
Background
A whole blood viscoelasticity detection technology invented by Hartert in 1948, the nature of viscoelasticity detection is recording resistance or friction force, dynamic change of blood coagulation is reflected by simulating blood viscoelasticity change of a whole blood specimen in a human body environment, and the process from activation of coagulation factors to formation of platelet-fibrin and platelet aggregation, formation of stable blood clot and fibrinolysis is monitored by using a physical principle, so that the rate, strength and stability (fibrinolysis level) of blood clot formation are reflected, and the whole process of coagulation and fibrinolysis is functionally evaluated. The whole blood coagulation process of all blood components except the vascular factors can be continuously reflected in real time, so that the risk of bleeding and thrombus of a patient can be judged. The method can more sensitively and comprehensively evaluate the abnormal state of blood coagulation, predicts the bleeding and death risk of the patient, is favorable for clinicians to clearly and definitely formulate blood transfusion strategies, guide reasonable medication, antithrombotic and other clinical treatments, and can effectively control the death rate of the patient.
Heparin is used as an anticoagulant and has good anticoagulation effect in vivo and in vitro. Heparin is capable of binding to Antithrombin (AT), and catalytically inactivating coagulation factors IIa, xa, IXa, XIa and XIIa. Under the condition that heparin does not exist, the AT inactivates the blood coagulation factor very slowly, heparin can be combined with the lysine part of the AT, so that the conformation of an arginine reaction center of antithrombin is changed, the AT is changed from a chronic thrombin inhibitor to a rapid inhibitor, and the speed of inactivating the blood coagulation factor is increased by 1000-2000 times. Heparin intervention significantly extends the time of parameter R in the thromboelastography. Heparanase is a kind of lyase capable of degrading heparin, and heparin can be degraded by adding heparanase into heparin-containing sample, and the blood coagulation time R is recovered to be normal.
Prior art CN201711158201.4 discloses a heparinase cup comprising: cup and heparanase lyophilized preparation. Also disclosed is a method of lyophilizing a formulation of a heparanase, comprising: and (3) sequentially performing pre-freezing treatment, sublimation treatment and drying treatment on the aqueous solution containing the heparinase, the protective agent, the buffer salt and the preservative in a predetermined proportion so as to obtain the heparinase freeze-dried preparation.
The prior art CN201811632170.6 discloses a heparin detection kit and application thereof, the heparin detection kit comprises a heparin detection reagent, the heparin detection reagent comprises heparinase I, the heparin detection reagent is stored in a solid state after being freeze-dried for long-term storage, and a certain amount of anhydrous calcium chloride, glycerol and D-trehalose are added into the heparinase I to be used as a protective agent, so that the detection reagent can exist stably for a long time, and the requirement of long-term storage is met.
In the prior art, the heparanase activator and the calcium chloride are respectively packaged into two freezing pipes, so that the liquid quantity is small, the adhesion is in the freezing pipes, the stability is poor, and the operation is complex; in order to solve the problems, the invention provides the thrombi elastography heparinase cup, which is convenient to detect, the sample and the activator are mixed more fully, the freeze-dried balls are longer in storage life, the stability is stronger and other reagent performances, the accuracy of clinical sample detection is greatly improved, and meanwhile, the problems of short quality guarantee period, incomplete reagent redissolution, dry liquid reagent and the like of the existing similar reagents are effectively solved.
Disclosure of Invention
The invention firstly provides a heparinase cup with a thrombus elasticity map, which comprises a reagent card, wherein the reagent card comprises a sample bin, a microfluidic channel, a quantifying chamber, an activator channel, a detection channel and a waste liquid bin, the sample bin is communicated with the microfluidic channel, the microfluidic channel is communicated with the quantifying chamber, and the quantifying chamber is communicated with the waste liquid bin; the quantifying chamber is communicated with the activator channel and the detection channel, and the microfluidic channel is communicated with the three channels;
the three channels are respectively provided with freeze-dried balls coated with an activating agent and freeze-dried balls coated with calcium chloride; comprises a heparanase coated freeze-dried ball and a calcium chloride coated freeze-dried ball; and an empty channel;
optionally, the lyoprotectant is contained in the lyoprotectant, and the lyoprotectant comprises mannitol, PEG8000, trehalose, dextran, bovine serum albumin, proclin300.
Preferably, the reagent card further comprises a sample cup.
Preferably, the freeze-dried balls adopt a liquid nitrogen pre-freezing technology, the activator, the heparinase and the calcium chloride are respectively freeze-dried and stored in a reagent card chip, and a vacuum aluminum foil bag is sealed in a double-layer mode.
Preferably, the vacuum aluminum foil bag material is two or three of PP, PET, AL, CPE, NY.
Preferably, the diameter of the freeze-dried balls is 1-4mm.
Preferably, the freeze-dried balls are packaged in chips and directly subjected to high-temperature hot-press welding, the temperature is 100-150 ℃, and the hot-press welding time is 1-20s.
Preferably, the activator comprises one or more of ellagic acid, kaolin, diatomite and kaolin;
optionally, the activator is formulated at a final concentration of 0.01-0.05mg/ml by mass volume;
optionally, the heparanase is heparanase I, and the final concentration of the prepared mass volume of the heparanase I is 0.5-5IU/ml;
optionally, the molar volume ratio of calcium chloride is 0.2M.
Preferably, the protective agent comprises mannitol 15%, PEG8000 3%, trehalose 6%, dextran 2%, bovine serum albumin 1%, proclin 300.05%.
The invention also provides a preparation method of the heparinase cup with the thrombus elasticity map, which comprises the following steps:
(1) Preparing freeze-dried balls coated with activator, freeze-dried balls coated with calcium chloride and freeze-dried balls coated with heparinase;
(2) And assembling the freeze-dried ball and the reagent card.
The invention also provides application of the heparinase cup with the thrombus elastography, wherein the application comprises a) preparation of a detection reagent or a kit; or b) a method of detecting vascular occlusive disease, optionally for non-diagnostic or non-therapeutic purposes.
Compared with the prior art, the invention has at least the following beneficial effects:
1. the freeze-dried ball prepared by the preparation process of the heparinase cup freeze-dried ball provided by the invention has the appearance: spherical, round and smooth.
2. The freeze-dried balls prepared by the preparation process of the heparinase cup freeze-dried balls provided by the invention have low water content and good re-solubility, so that the stability of the concentration of the freeze-dried balls is prevented from being influenced by high water content, and the stability of the performance of the freeze-dried balls is improved.
3. The preparation of the heparinase cup freeze-dried ball provided by the invention effectively solves the problems of short storage period and the like of the conventional similar reagent in cold chain transportation and low-temperature preservation.
4. The detection is completed by adopting a fully-closed matched instrument, the whole blood can be directly operated by inserting the collected anticoagulated venous whole blood collection tube of sodium citrate into a reagent card during the detection, the complex procedures such as manual sample adding and the like are not needed, the result can be displayed by waiting for 30min, the operation is simple and convenient, and the detection is rapid;
5. meanwhile, the problem that the inner surface of the sample cup is adhered to and falls off from the blood clot in the common detection of the thrombus elastography in the detection process is solved, the accuracy and the stability of the common coagulation detection of the thrombus elastography are improved, and a reliable basis is provided for clinical diagnosis.
Drawings
FIG. 1 is a schematic diagram of the structure of a thromboelastography heparinase cup reagent card;
FIG. 2 is a photograph of a heparinase cup freeze-dried ball of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Example 1A thromboelastography heparinase cup
1. A thrombus elastography heparinase cup, which comprises a reagent card, wherein the structure of the reagent card is shown in fig. 1, the reagent card comprises a sample bin, a micro-flow channel, a quantifying chamber, an activator channel, a detecting channel and a waste liquid bin, the sample bin is communicated with the micro-flow channel, the micro-flow channel is communicated with the quantifying chamber, and the quantifying chamber is communicated with the waste liquid bin; the quantifying chamber is communicated with the activator channel and the detection channel, and the microfluidic channel is communicated with the three channels;
the three channels are respectively provided with freeze-dried balls coated with an activating agent and freeze-dried balls coated with calcium chloride; comprises a heparanase coated freeze-dried ball and a calcium chloride coated freeze-dried ball; and an empty channel.
Example 2 procedure for lyophilization
1. S1 (ellagic acid reagent), R1 (heparinase I reagent), S2 (calcium chloride reagent) and lyoprotectant to be lyophilized are formulated according to the concentrations of ellagic acid, heparinase I, calcium chloride and lyoprotectant: mannitol 15%, PEG8000 3%, trehalose 6%, dextran 2%, bovine serum albumin 1%, proclin 300.05%.
2. Lyophilization parameter information:
2.1, freezing the prepared S1, R1 and S2 into pellets with the volume of 5-15uL by using a liquid nitrogen pre-freezing technology, and transferring the pellets into a freeze dryer;
table 1 procedure for lyophilization
The freeze dryer can be selected as the model: pilot10-15ES, manufacturer is Boyi kang (Beijing) instruments Co., ltd.
Example 3 detection of heparinase cup reagent card Using a Thrombus elastography
1. The whole blood sample to be detected enters the reagent card through the sample bin, finally flows into the detection channel along the microfluidic channel, freeze-dried reagents in the first channel and the second microfluidic channel are dissolved in the sample, and the detection is carried out by matching with the ST-1000 full-automatic thromboelastography instrument, wherein the detection method comprises the following steps:
1.1, collecting at least 2mL of venous whole blood in a sodium citrate anticoagulation blood collection tube;
1.2 samples collected should be stored at room temperature, and before detection, the blood collection tube should be gently inverted at least 5 times to ensure thorough mixing of samples, and samples with obvious clotting should not be used.
1.3 taking out the reagent card from the vacuum aluminum foil bag, holding the reagent card by hand, and pulling out the protective sheath of the needle on the reagent card.
1.4 gently inverting the blood collection tube containing venous whole blood for at least 5 times, mixing the samples uniformly, inserting the mixed samples into the needle hole of the reagent card, and enabling the rubber plug of the blood collection tube to face downwards so as to puncture the needle thoroughly.
1.5, inserting the reagent card into the instrument detection groove according to the channel using popup window prompt of the software, and automatically completing the reagent card detection by the full-automatic thromboelastography instrument after the screen prompts that the test item of the patient is matched with the reagent card without error, clicking confirmation and jumping the experimental interface.
1.6 clotting time R ends at about 1-5min, if the complete fibrinolysis process is observed for a period of up to 30min.
Example 4 accuracy detection of thromboelastography heparinase cup detection reagent
1.6 fresh whole blood samples without heparin residues with different blood coagulation speeds are selected, the samples are respectively measured by using the reagent card prepared by the invention and the marketed thromboelastography kit (heparinase cup), and the obtained results are compared.
2. Wherein, thromboelastography (heparinase cup) detection kit (control reagent): s1 is a kaolin reagent, S2 is a CaCl2 reagent and a sample cup, the manufacturer is Chongqing Dingrun medical instrument Limited liability company, and the kit batch number is 20221201. The contrast agent is operated by adopting the flow corresponding to the kit.
3. For the thromboelastography heparinase cup reagent card obtained above, the test procedure was tested as described above.
4. The blood coagulation time R value was measured and the results of 6 samples are shown in the following table. Where R (min) refers to the time (min) required for blood to begin to form a first fibrin clot (amplitude of the trace up to 2 mm) in a thromboelastometer. R1-R2 is less than 1.0min, which indicates that no heparin residue exists in the sample; wherein R1 is the clotting time of the ordinary cup test, and R2 is the clotting time of the heparinase cup test.
From the following table, it can be seen that the R1-R2 of the reagent and the comparative reagent have significant differences, the detection results of the reagent R1-R2 all indicate that no heparin residue exists in the sample, and the detection results of the comparative reagent have individual heparin residues in the display sample, which indicates that the reagent has high preparation degree and better sensitivity.
Table 2 accuracy testing of thromboelastography heparinase cup test reagents
Example 5 test of thromboelastography heparinase cup detection reagent
1. Respectively 2 ℃, 5 ℃, 8 ℃, 11 ℃, 14 ℃, 17 ℃, 20 ℃, 23 ℃, 25 ℃, taking the quality control level 1 and the quality control level 2, and performing a thrombus elastography test by using a thrombus elastography heparinase cup detection reagent. Wherein R1 is the clotting time of the ordinary cup test, and R2 is the clotting time of the heparinase cup test.
2. Accelerating the materials for 0, 2, 4, 6, 8, 10, 12 and 15 days in a 37 ℃ incubator, taking a quality control level of 1 (0 min < blood coagulation time (R) is less than or equal to 2.0min;0min < blood clot formation time (K) is less than or equal to 5.0min;60 DEG < blood coagulation rate (Angle) <90 DEG; 10.00mm < blood clot strength (MA) is less than or equal to 50.00mm; main component: sodium citrate anticoagulated pig plasma freeze-dried powder: promaide (North) technology Co., ltd.), quality control II (2.0 min < blood coagulation time (R), 0min < blood clot formation time (K) is less than or equal to 5.0min;60 DEG < blood coagulation rate (Angle) <90 DEG; 50.00mm < blood clot strength (MA) is less than or equal to 90.00mm; main component: sodium citrate anticoagulated pig plasma freeze-dried powder: primed (North) technology Co., ltd.), and carrying out a thromboelastase cup detection reagent. Wherein R1 is the clotting time of the ordinary cup test, and R2 is the clotting time of the heparinase cup test.
3. For the thromboelastography heparinase cup reagent card obtained above, the test procedure was tested as described above.
4. From the following table, it can be seen that the test R value of the reagent is within 10% after acceleration for 15 days at 2 ℃, 5 ℃, 8 ℃, 11 ℃, 14 ℃, 17 ℃, 20 ℃, 23 ℃, 25 ℃ and 37 ℃, and the result deviation and precision test are all within 10%, and the acceleration stability is good. The reagent has the storage temperature of 2-25 ℃, can solve the problem of low-temperature storage and cold chain transportation, and is superior to the available period of the existing reagent in the market.
TABLE 3 quality control level 1 thromboelastography test of different temperatures of heparinase cup detection reagents
TABLE 4 quality control level 2 thromboelastography test of different temperatures of heparinase cup detection reagents
TABLE 5 accelerated test of thromboelastography heparinase cup detection reagent for quality control level 1
TABLE 6 accelerated test of thromboelastography heparinase cup detection reagent for quality control level 2
Example 6 Long term stability test of thromboelastography heparinase cup detection reagent
1. And respectively storing for 0, 3, 6, 9, 12, 15, 18, 21, 24, 36 and 48 months at the temperature of 2-25 ℃, taking the same batch of quality control level 1 and quality control level 2, and testing the thrombus elastography by using the experimental reagent. Wherein R1 is the clotting time of the ordinary cup test, and R2 is the clotting time of the heparinase cup test.
2. For the thromboelastography heparinase cup reagent card obtained above, the test procedure was tested as described above.
3. From the table below, it can be seen that the test R value of the reagent is within 10% after being stored for 48 months at 2-25 ℃, which indicates that the reagent has good long-term stability and is superior to the validity stability of the existing reagents in the market.
TABLE 7 quality control level 1 stability test of thromboelastography heparinase cup detection reagents
Table 8 quality control level 2 stability test of thromboelastography heparinase cup assay reagents
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A thromboelastography heparinase cup, characterized in that it comprises a reagent card comprising a sample compartment, a microfluidic channel, a quantifying chamber, an activator channel, a detection channel and a waste liquid compartment, wherein the sample compartment is communicated with the microfluidic channel, the microfluidic channel is communicated with the quantifying chamber, and the quantifying chamber is communicated with the waste liquid compartment; the quantifying chamber is communicated with the activator channel and the detection channel, and the microfluidic channel is communicated with the three channels;
the three channels are respectively provided with freeze-dried balls coated with an activating agent and freeze-dried balls coated with calcium chloride; comprises a heparanase coated freeze-dried ball and a calcium chloride coated freeze-dried ball; and an empty channel;
optionally, the lyoprotectant is contained in the lyoprotectant, and the lyoprotectant comprises mannitol, PEG8000, trehalose, dextran, bovine serum albumin, proclin300.
2. The thromboelastography heparinase cup according to claim 1, wherein said reagent card further comprises a sample cup.
3. The thromboelastography heparinase cup according to claim 1, wherein said freeze-dried balls are freeze-dried by liquid nitrogen pre-freezing technique, and said activator, heparinase and calcium chloride are stored in reagent card chip and vacuum aluminum foil bag is sealed in double layers.
4. A thrombelastogram heparinase cup according to claim 3 wherein said vacuum aluminium foil bag material is two or three of PP, PET, AL, CPE, NY.
5. A thrombi elastography heparinase cup according to claim 3 wherein said freeze dried balls are 1-4mm in diameter.
6. A thrombi elastography heparinase cup according to claim 3 wherein said lyophilized pellet is packaged in a chip directly and heat-pressed at 100-150 ℃ for 1-20s.
7. The thromboelastography heparinase cup according to claim 1, wherein said activator comprises one or a mixture of several of ellagic acid, kaolin, diatomaceous earth, kaolin;
optionally, the activator is formulated at a final concentration of 0.01-0.05mg/ml by mass volume;
optionally, the heparanase is heparanase I, and the final concentration of the prepared mass volume of the heparanase I is 0.5-5IU/ml;
optionally, the molar volume ratio of calcium chloride is 0.2M.
8. The thromboelastography heparinase cup according to claim 1, characterized in that said protective agent comprises mannitol 15%, PEG8000 3%, trehalose 6%, dextran 2%, bovine serum albumin 1%, proclin 300.05%.
9. A method of preparing a thromboelastography heparinase cup according to any one of claims 1-8, characterised in that it comprises the steps of: (1) Preparing freeze-dried balls coated with activator, freeze-dried balls coated with calcium chloride and freeze-dried balls coated with heparinase;
(2) And assembling the freeze-dried ball and the reagent card.
10. Use of a thromboelastography heparinase cup according to any of the claims 1-8, characterised in that said use is a) the preparation of a detection reagent or kit; or b) a method of detecting vascular occlusive disease, optionally for non-diagnostic or non-therapeutic purposes.
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