CN112841171A - Preparation method and application of anticoagulated pig blood and pig plasma used in thrombus test - Google Patents
Preparation method and application of anticoagulated pig blood and pig plasma used in thrombus test Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
本发明涉及实验药剂技术领域，公开了一种用于血栓试验中的抗凝猪血、猪血浆的制备方法及应用，本发明所制得的抗凝猪血及抗凝富血小板猪血浆能够代替人血进行血栓试验，模拟人出血和止血的情况，使血栓试验能够顺利进行，降低试验成本，有利于节省人血资源，同时还有利于血栓弹力图仪、凝血仪的仪器研发、性能测试以及一些临床药物的开发。
The invention relates to the technical field of experimental agents, and discloses a preparation method and application of anticoagulated pig blood and pig plasma used in thrombus test. The anticoagulated pig blood and anticoagulated platelet-rich pig plasma prepared by the invention can replace the The thrombus test on human blood simulates the situation of human hemorrhage and hemostasis, so that the thrombus test can be carried out smoothly, reduce the test cost, save human blood resources, and also facilitate the development, performance testing and performance testing of thromboelastography and coagulation instruments. Development of some clinical drugs.
Description
Technical Field
The invention belongs to the technical field of experimental medicaments, and particularly relates to a preparation method and application of anticoagulated pig blood and pig plasma used in a thrombus test.
Background
Because of the research, development and preparation of thromboelastogram instruments and thrombometers, and the development of clinical drugs for diseases (such as apoplexy, epilepsy, myocardial infarction, uremia and the like), thrombosis tests by anticoagulated blood and anticoagulated plasma are required.
Among other things, thromboelastography is used to monitor the coagulation status of blood samples to aid in clinical evaluation of patients. The thromboelastogram instrument is widely applied to monitoring postoperative bleeding and/or thrombosis in or at the later stage of cardiac surgery, organ transplantation, tumor and radiotherapy, assists a clinician to accurately judge the coagulation condition of a clinical patient through monitoring and analyzing the coagulation process of a blood sample, and predicts the clinical condition according to the coagulation evaluation result.
A thrombometer is an instrument that performs laboratory examinations of thrombus and hemostasis. The detection indexes of the hemostasis and thrombosis molecular markers are closely related to clinical diseases such as atherosclerosis, cardiovascular and cerebrovascular diseases, diabetes and arteriovenous thrombosis, and the theory and treatment work research of the traditional Chinese medicine on promoting blood circulation and removing blood stasis also relate to the hemostasis and thrombosis problems.
But because human blood is lack, expensive, high in biological safety risk and strict in supervision, thrombus tests are difficult to perform, the cost is high, and the development and performance tests of instruments of thromboelastography and thrombometers and the development of some clinical medicines are severely limited.
Disclosure of Invention
In order to solve the above problems, the first objective of the present invention is to provide a method for preparing anticoagulated pig blood for thrombus test and its application, wherein the anticoagulated pig blood can replace human blood to perform thrombus test.
The invention also aims to provide a preparation method and application of the anticoagulation porcine plasma for thrombus test, and the prepared anticoagulation platelet-rich porcine plasma can replace human plasma to carry out thrombus test.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a preparation method of anticoagulated pig blood for a thrombus test, which comprises the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 2-4 hours;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in the S2, the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
In the invention, compared with the preparation of other existing blood, the preparation of anticoagulated pig blood is more rigorous, the anticoagulated pig blood can only be prepared by sodium citrate solution, the concentration of the sodium citrate solution cannot be too high or too low, the span cannot be too large, the concentration of the sodium citrate solution is within the range of 3.2-3.8%, and the sodium citrate solution is mixed with the pig blood in the weight ratio of 1:9, the effect is best, and the anticoagulated pig blood can be used for thrombus test; the anticoagulation pig blood prepared by the preparation method can replace human blood to carry out thrombus test, simulate the conditions of human bleeding and hemostasis, ensure that the thrombus test can be smoothly carried out, reduce the test cost, be conductive to saving human blood resources, and be conductive to the research and development of instruments, performance test and development of some clinical drugs of thromboelastography instruments and thrombometers.
Further, in order to obtain a better anticoagulation effect of pig blood, the concentration of the sodium citrate solution is preferably 3.8% when the pig blood is shaken up and then left to stand for 3 hours in S2.
Furthermore, in order to make the prepared anticoagulation pig blood have better effect, the hematocrit of the anticoagulation pig blood is 29-35 percent; the total protein of the anticoagulated pig blood is 55g/L-65 g/L.
Further, storing the anticoagulated pig blood in an environment of 2-8 ℃ for 30 days; the anticoagulated pig blood is used for a thrombus test in a valid period.
The invention also provides application of the anticoagulation pig blood prepared by the preparation method in a thrombus test, which can replace human blood to carry out the thrombus test, simulate the conditions of human bleeding and hemostasis and facilitate the research and development of hemostasis effect.
The invention also provides a preparation method of the anticoagulation pig plasma used in the thrombus test, which comprises the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 700g-1220g, and the centrifugal time is 5-10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In the invention, the anticoagulation platelet-rich pig plasma prepared by the preparation method has rich platelet content, the centrifugation condition is limited to the centrifugal force of 700g-1220g, the centrifugation time is 5-10min, and the loss degree of the platelets can be controlled, so that the subsequent thrombosis test is facilitated, and the blood coagulation caused by a series of reactions of the platelets is facilitated, thereby the research and development of the hemostasis effect are carried out.
Wherein, the platelet has the main function of blood coagulation in human body; can effectively stop bleeding at ordinary times, and the blood of the organism can not be lost too much due to the contribution of the blood platelet and the thrombin. Therefore, the platelet and thrombin contents are of critical importance in thrombus testing.
Further, in S2, the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed in a weight ratio of 1: 9. In the invention, the anticoagulant can only adopt a sodium citrate solution, the concentration of the sodium citrate solution cannot be too high or too low, the span cannot be too large, the concentration of the sodium citrate solution is within the range of 3.2-3.8%, and the sodium citrate solution is mixed with the pig blood in the weight ratio of 1:9, so that the best effect is achieved.
Further, in S3, the centrifugation treatment is carried out under the temperature condition of 20-24 ℃ and the centrifugal force of 1220g for 5 minutes; or centrifuging at 20-24 deg.C under centrifugal force of 700g for 10 min. In the invention, the loss degree of the platelets can be better controlled through the limitation of the conditions, so that the subsequent thrombus test is facilitated, and the development of the hemostatic effect is facilitated due to the coagulation caused by a series of reactions of the platelets.
Further, storing the anticoagulated platelet-rich pig plasma in an environment of 20-24 ℃ for 22 days; the reason is that platelets are not easily subjected to morphological changes at (22. + -. 2). degree.C, and easily retain their physiological activities. Because the loss of the blood platelets after cryopreservation is large, the in vivo survival rate is low, and the hemostatic effect is only about 55% of that of fresh blood platelets after the frozen blood platelets are thawed and washed, the blood platelets need to be frozen, and a cryoprotectant is added: dimethyl sulfoxide (DMSO) and glycerol. The anticoagulant platelet-rich pig plasma is stored in an environment with the temperature of-20 ℃, the period of validity is 1 year, the plasma is unfrozen in the later use, washing is not needed, or a small amount of plasma is used for dilution, the in vitro recovery rate can reach 90 percent, the plasma is similar to fresh platelets in blood circulation, and the anticoagulant platelet-rich pig plasma is used for thrombus tests.
The invention also provides an application of the anticoagulation platelet-rich pig plasma prepared by the preparation method in a thrombus test.
The invention has the beneficial effects that: compared with the prior art, the anticoagulated pig blood and the anticoagulated platelet-rich pig plasma prepared by the method can replace human blood to carry out thrombus test, simulate the condition of human bleeding and hemostasis, ensure that the thrombus test can be smoothly carried out, reduce the test cost, be beneficial to saving human blood resources, and be beneficial to the research and development of instruments, performance test and development of some clinical drugs of thromboelastography instruments and thrombometers.
Drawings
FIG. 1 shows a flow chart of a preparation method of anticoagulated pig blood and anticoagulated pig plasma.
FIG. 2 shows the results of thromboelastography of the anticoagulated platelet-rich porcine plasma obtained in example 4.
FIG. 3 shows the results of thromboelastography of the anticoagulated platelet-rich porcine plasma obtained in example 5.
FIG. 4 shows the results of thromboelastography of the anticoagulated platelet-rich porcine plasma obtained in example 6.
FIG. 5 shows the results of thromboelastography of the anticoagulated platelet-rich porcine plasma obtained in example 7.
FIG. 6 shows the platelets observed under an electron microscope from the anticoagulated platelet-rich porcine plasma obtained in example 1.
FIG. 7 shows the platelets observed under an electron microscope from the anticoagulated platelet-rich porcine plasma obtained in example 2.
FIG. 8 shows the erythrocytes observed under an electron microscope of the anticoagulated pig blood prepared in example 1.
FIG. 9 shows the erythrocytes observed under an electron microscope of the anticoagulated pig blood prepared in example 2.
FIG. 10 shows the erythrocytes observed under an electron microscope from the anticoagulated pig blood prepared in example 3.
FIG. 11 shows a glass plate used for anticoagulated pig blood observed under an electron microscope.
FIG. 12 is a graph showing the aggregation of erythrocytes from platelet-rich anticoagulated porcine plasma after addition of calcium ions.
FIG. 13 shows a magnified image of a portion of the red blood cell aggregates.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preparation method of anticoagulated pig blood for a thrombus test, which comprises the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 2-4 hours; the anticoagulant plays a role in anticoagulation, and is shaken up in time after blood collection so that the anticoagulant is fully contacted with the whole blood;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in the S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9. Sodium citrate can inactivate calcium and prevent blood coagulation.
In the invention, compared with the preparation of other existing blood, the preparation of anticoagulated pig blood is more rigorous, the anticoagulated pig blood can only be prepared by sodium citrate solution, the concentration of the sodium citrate solution cannot be too high or too low, the span cannot be too large, the concentration of the sodium citrate solution is within the range of 3.2-3.8%, and the sodium citrate solution is mixed with the pig blood in the weight ratio of 1:9, the effect is best, and the anticoagulated pig blood can be used for thrombus test; the anticoagulation pig blood prepared by the preparation method can replace human blood to carry out thrombus test, simulate the conditions of human bleeding and hemostasis, ensure that the thrombus test can be smoothly carried out, reduce the test cost, be conductive to saving human blood resources, and be conductive to the research and development of instruments, performance test and development of some clinical drugs of thromboelastography instruments and thrombometers.
Specifically, collecting pig blood by jugular arteriovenous blood collection method;
fixing the pig, and shearing off the hair of the neck; then pressing the jugular vein of the pig, expanding the blood vessel to determine the position of the lower needle, and disinfecting the position by using 75% alcohol; taking a 5 or 10ml syringe, holding the syringe with a needle by an experimenter, sucking the anticoagulant with the defined proportion and concentration, then pricking the needle at the position of the blood vessel bulge, and automatically introducing blood into the syringe. Generally, an adult pig can take 250-500ml blood, and the blood drawing is necessarily slow. Hemostasis by compression is required after the collection.
Specifically, in order to obtain a better anticoagulation effect of pig blood, in S2, the solution is shaken and then left to stand for 3 hours, and the concentration of the sodium citrate solution is preferably 3.8%.
Specifically, in order to make the prepared anticoagulated pig blood have better effect, the hematocrit of the anticoagulated pig blood is 29-35%, and if the hematocrit comes in and goes out, self-regulation is carried out; the total protein of the anticoagulated pig blood is 55g/L-65g/L, and if the total protein goes in and out, the automatic regulation is carried out.
Specifically, the anticoagulated pig blood is stored in an environment of 2-8 ℃ for 30 days; anticoagulated pig blood was used for the thrombosis test.
The invention also provides application of the anticoagulation pig blood prepared by the preparation method in a thrombus test.
The invention also provides a preparation method of the anticoagulation pig plasma used in the thrombus test, which comprises the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up; shaking up in time after blood collection to ensure that the anticoagulant is fully contacted with the whole blood;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 700g-1220g, and the centrifugal time is 5-10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In the invention, the anticoagulation platelet-rich pig plasma prepared by the preparation method has rich platelet content, the centrifugation condition is limited to the centrifugal force of 700g-1220g, the centrifugation time is 5-10min, and the loss degree of the platelets can be controlled, so that the subsequent thrombosis test is facilitated, and the blood coagulation caused by a series of reactions of the platelets is facilitated, thereby the research and development of the hemostasis effect are carried out.
Wherein, the platelet has the main function of blood coagulation in human body; can effectively stop bleeding at ordinary times, and the blood of the organism can not be lost too much due to the contribution of the blood platelet and the thrombin. Therefore, the platelet and thrombin contents are of critical importance in thrombus testing.
The platelet is a blood cell with the lightest specific gravity in the visible components of the blood, and a thicker and purer platelet product can be separated and extracted from the whole blood by a centrifugal method by utilizing larger specific gravity difference.
The raw material whole blood for preparing the anticoagulant platelet-rich pig plasma needs to be collected by adopting a multi-connected bag, and then the whole blood is stored and transported within 6 hours at room temperature.
Specifically, in S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9. In the invention, the anticoagulant can only adopt sodium citrate solution, the concentration of the sodium citrate solution cannot be too high or too low, the span cannot be too large, the concentration of the sodium citrate solution is within the range of 3.2-3.8%, and the sodium citrate solution is mixed with the pig blood in the weight ratio of 1:9, so that the best effect is achieved.
Specifically, in S3, the centrifugation treatment specifically includes:
s31, under the temperature condition of 20-24 ℃, the centrifugal force is 1220g, and the centrifugation is 5 minutes; or the centrifugal force is 700g, and the centrifugation is carried out for 10 minutes; allowing erythrocyte to sink, and keeping most of platelets in plasma due to small specific gravity to obtain platelet-rich plasma (PRP);
s32, platelet rich plasma separation transfer:
(1) taking out blood from the centrifuge cup, avoiding vibration, hanging the blood on a plasma separating rack, and standing for more than 30 minutes; binding the blood distribution catheter to the first transfer empty bag by using a rubber ring; a plastic clip is placed on the transfer tube of the second transfer empty bag.
(2) Visually judging whether the plasma and the red blood cells are clearly layered, whether the plasma and the red blood cells are hemolyzed, have bubbles, severe chyle and abnormal color, and whether blood bags and pipelines are intact; and breaking off the blood distribution catheter fixed on the first transfer empty bag if the blood distribution catheter meets the requirements.
(3) Placing the blood on a plasma separating clamp to separate platelet-rich plasma into a first transfer empty bag; hemostatic forceps are arranged on the rich board pulp bag pipeline; the hemostat is loosened, and the air in the plasma bag is discharged into the erythrocyte bag.
(4) Suspending the red blood cells; breaking the erythrocyte preservation solution blocking tube, transferring the erythrocyte preservation solution into the erythrocyte bag, stopping the blood forceps at about 1/3 of the outlet tube of the erythrocyte bag, and slightly shaking to uniformly mix the erythrocyte and the preservation solution.
(5) And adding a plastic clip at the outlet of the platelet-rich plasma bag.
(6) And (6) thermally sealing the conduit.
Wherein, the anticoagulant platelet-rich pig plasma is stored in an environment of 20-24 ℃ with the validity period of 22 days; the reason is that platelets are not easily subjected to morphological changes at (22. + -. 2). degree.C, and easily retain their physiological activities. Because the loss of the blood platelets after cryopreservation is large, the in vivo survival rate is low, and the hemostatic effect is only about 55% of that of fresh blood platelets after the frozen blood platelets are thawed and washed, the blood platelets need to be frozen, and a cryoprotectant is added: dimethyl sulfoxide (DMSO) and glycerol. The anticoagulant platelet-rich pig plasma is stored in an environment with the temperature of-20 ℃, the validity period is 1 year, the anticoagulant platelet-rich pig plasma is unfrozen in later use without washing or diluted by a small amount of plasma, the in vitro recovery rate can reach 90 percent, the anticoagulant platelet-rich pig plasma is similar to fresh platelets in blood circulation, and the anticoagulant platelet-rich pig plasma is used for thrombus tests.
The invention also provides an application of the anticoagulation platelet-rich pig plasma prepared by the preparation method in a thrombus test.
The preparation method and application of the anticoagulated pig blood and pig plasma for thrombosis test according to the present invention will be specifically described below with reference to specific examples.
Example 1
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 3 hours;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
The obtained anticoagulated pig blood has hematocrit of 29-35% and total protein of 55-65 g/L; storing the prepared anticoagulated pig blood in an environment of 2-8 ℃ for 30 days; anticoagulated pig blood was used for the thrombus test during the expiration period.
The anticoagulated pig blood prepared in example 1 is observed, and the integrity of erythrocytes inside the anticoagulated pig blood is observed, as shown in fig. 8, which indicates that the anticoagulated pig blood prepared in example 1 is not hemolyzed, i.e. is well controlled by the above preparation process, so that the subsequent thrombosis test is convenient, and the conditions of human bleeding and hemostasis are simulated.
Example 2
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 2 hours;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.2%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
The obtained anticoagulated pig blood has hematocrit of 29-35% and total protein of 55-65 g/L; storing the prepared anticoagulated pig blood in an environment of 2-8 ℃ for 30 days; anticoagulated pig blood was used for the thrombus test during the expiration period.
The anticoagulated pig blood prepared in example 1 is observed, and the integrity of erythrocytes inside the anticoagulated pig blood is observed, as shown in fig. 9, which indicates that the anticoagulated pig blood prepared in example 1 is not hemolyzed, i.e. is well controlled by the above preparation process, so that the subsequent thrombosis test is convenient, and the conditions of human bleeding and hemostasis are simulated.
Example 3
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 4 hours;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.5%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
The obtained anticoagulated pig blood has hematocrit of 29-35% and total protein of 55-65 g/L; storing the prepared anticoagulated pig blood in an environment of 2-8 ℃ for 30 days; anticoagulated pig blood was used for the thrombus test during the expiration period.
The anticoagulated pig blood prepared in example 1 is observed, and the integrity of erythrocytes inside the anticoagulated pig blood is observed, as shown in fig. 10, which indicates that the anticoagulated pig blood prepared in example 1 is not hemolyzed, i.e. is well controlled by the above preparation process, so that the subsequent thrombosis test is convenient, and the conditions of human bleeding and hemostasis are simulated.
Example 4
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 700g, and the centrifugal time is 10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Storing the prepared anticoagulant platelet-rich pig plasma in an environment of 22 +/-2 ℃ for 22 days; anticoagulant platelet-rich porcine plasma is used for thrombus test in the validity period.
The anticoagulated platelet-rich porcine plasma obtained in example 4 was 75.1 as shown in FIG. 2, as measured by a thromboelastography.
Example 5
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 716g, and the centrifugal time is 10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.2%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Storing the prepared anticoagulant platelet-rich pig plasma in an environment of 22 +/-2 ℃ for 22 days; anticoagulant platelet-rich porcine plasma is used for thrombus test in the validity period.
The anticoagulated platelet-rich porcine plasma obtained in example 5 was 75.7 as shown in FIG. 3.
Example 6
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 1220g, and the centrifugal time is 5 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Storing the prepared anticoagulant platelet-rich pig plasma in an environment of 22 +/-2 ℃ for 22 days; anticoagulant platelet-rich porcine plasma is used for thrombus test in the validity period.
The anticoagulated platelet-rich porcine plasma obtained in example 6 was 68.1 as shown in FIG. 4.
Example 7
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 1220g, and the centrifugal time is 5 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.2%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Storing the prepared anticoagulant platelet-rich pig plasma in an environment of 22 +/-2 ℃ for 22 days; anticoagulant platelet-rich porcine plasma is used for thrombus test in the validity period.
The anticoagulated platelet-rich porcine plasma obtained in example 7 was 69.4 as shown in FIG. 5.
Referring to fig. 2-5, the MA values of the anticoagulated platelet-rich porcine plasma prepared in examples 4-7 are all within the range of 68-76, while the MA values of the porcine plasma used in the prior art are generally only 40, and the highest MA value is only 55-60, while the anticoagulated platelet-rich porcine plasma prepared by the preparation method of the present invention can reach 68-76, which is far higher than the MA values in the prior art.
Referring to FIG. 6, the platelet-rich porcine plasma obtained in example 1 is shown as platelets observed under an electron microscope;
referring to FIG. 7, the platelet of the anticoagulant platelet-rich porcine plasma obtained in example 2 is shown under an electron microscope.
After calcium ions are added into the anticoagulant platelet-rich porcine plasma prepared in examples 4-7, erythrocytes begin to aggregate as shown in fig. 12-13, and then blood clots are formed, which play a role in hemostasis similar to blood vessels and facilitate the development of hemostasis effect.
Comparative example 1
S1, collecting pig blood in an aseptic environment;
s2, adding an anticoagulant into the pig blood, wherein the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 2%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
In comparison with the examples, in comparative example 1, since the concentration of the sodium citrate solution is too low, the blood just comes out without contacting with sufficient anticoagulant, self-forms coagulation, and cannot be made into anticoagulated blood or plasma.
Comparative example 2
S1, collecting pig blood in an aseptic environment;
s2, adding an anticoagulant into the pig blood, wherein the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 4%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Compared with the examples, in comparative example 1, because the concentration of the sodium citrate solution is too high, an osmotic pressure difference is formed, the osmotic pressure inside and outside the erythrocyte is different, and the erythrocyte is subjected to swelling rupture or shrinkage, so that hemolysis is caused.
Comparative example 3
S1, collecting pig blood in an aseptic environment;
s2, adding an anticoagulant into the pig blood, wherein the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 8.
Compared with the embodiment, in the comparative example 1, because the proportion of the sodium citrate solution is too low, the pig blood is coagulated without being mixed with the anticoagulant.
Comparative example 4
S1, collecting pig blood in an aseptic environment;
s2, adding an anticoagulant into the pig blood, wherein the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 10.
Compared with the examples, in comparative example 1, hemolysis is caused due to the high proportion of sodium citrate solution.
Comparative example 5
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 500g, and the centrifugal time is 10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Compared with example 4, the anticoagulated platelet-rich pig plasma prepared in comparative example 5 has a lower platelet content due to the excessively small centrifugal force during the centrifugation.
Comparative example 6
S1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 2000g, and the centrifugal time is 5 min;
s4, after completion of the centrifugation, rupture of erythrocytes was observed.
In S2, the anticoagulant is sodium citrate solution, the concentration of the sodium citrate solution is 3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
Compared with example 4, the anticoagulated platelet-rich porcine plasma obtained in comparative example 6 causes erythrocyte rupture due to excessive centrifugal force.
The platelet-rich anticoagulant platelet-rich porcine plasma obtained in examples 4-7 was used in the following experiments.
1. Platelet aggregation assay in platelet-rich plasma (PAgT)
The principle is as follows: turbidity method: adding human aggregation agent into Platelet Rich Plasma (PRP), platelet aggregation, plasma turbidity change and transmittance increase, and converting the turbidity change into electric signal and recording by platelet aggregation instrument to form platelet aggregation curve.
The extent and rate of platelet aggregation can be understood from the platelet aggregation curve. The resistance method (impedance method) is based on the principle of electrical impedance,
a method for measuring platelet aggregation of a whole blood sample by amplifying and recording a change in a minute current or impedance between electrode probes immersed in the whole blood sample.
2. Platelet release reaction product assay in platelet rich plasma
The principle is as follows: beta-thrombocyte globulin (B-thromboglobin, B-TG) and platelet fourth factor (platelet factor4, PF4) are proteins specific to platelet a granules. When excessive platelets are activated and the release reaction is accelerated in vivo, the plasma concentrations of both increase, and therefore, B-TG and PF4 are important indicators of platelet activation. ELISA method is mostly used for detecting the two. Coating a microporous plate with an antibody against B-TG or PF4, adding human plasma to be tested, combining B-TG or PF4 in the plasma with the coated antibody, adding an enzyme-labeled anti-beta-TG antibody or anti-PF 4 antibody, combining with B-TG or PF4 bound on the plate, and adding a human substrate for color development. The color intensity is proportional to the content of B-TG or PF4 in the plasma to be tested, and the content of B-TG or PF4 in the plasma to be tested is calculated from the standard curve.
3. Platelet-rich plasma Clot Retraction Test (CRT)
The principle is as follows: ca2+ and thrombin are added into the platelet-rich plasma to make the plasma coagulate to form clot, and platelet contractile protein makes the platelet stick out of the pseudopodia, and the front end of the pseudopodia is connected to the fibrin bundle. When the pseudopoda contracts centripetally, the fibrin mesh is reduced, and the volume of the precipitated serum is measured to reflect the contraction capacity of the platelet clot.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of anticoagulated pig blood used in a thrombus test is characterized by comprising the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood, shaking up and standing for 2-4 hours;
s3, shaking up again, and subpackaging to obtain anticoagulated pig blood;
in the S2, the anticoagulant is a sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed according to the weight ratio of 1: 9.
2. The method of claim 1, wherein the concentration of the sodium citrate solution in S2 after shaking and standing for 3 hours is 3.8%.
3. The method for preparing anticoagulated pig blood for thrombus test according to claim 1, wherein the anticoagulated pig blood has a hematocrit of 29% -35%; the total protein of the anticoagulated pig blood is 55g/L-65 g/L.
4. The method for preparing anticoagulated pig blood for thrombus test according to claim 3, wherein the anticoagulated pig blood is stored in an environment of 2-8 ℃.
5. Use of anticoagulated pig blood prepared by the method for preparing anticoagulated pig blood for thrombus test according to claims 1-4 in thrombus test.
6. A preparation method of anticoagulated pig plasma used in a thrombus test is characterized by comprising the following steps:
s1, collecting pig blood in an aseptic environment;
s2, adding the anticoagulant into the pig blood and shaking up;
s3, after the pig blood is cooled, carrying out centrifugal treatment, wherein the centrifugal force is 700g-1220g, and the centrifugal time is 5-10 min;
and S4, collecting supernatant after the centrifugation is finished, and obtaining the anticoagulant platelet-rich pig plasma.
7. The method for preparing anticoagulated pig plasma for thrombus test according to claim 6, wherein in S2, the anticoagulation agent is sodium citrate solution, the concentration of the sodium citrate solution is 3.2% -3.8%, and the sodium citrate solution and the pig blood are mixed in a weight ratio of 1: 9.
8. The method for preparing anticoagulated porcine plasma for thrombus test according to claim 6, wherein in S3, the centrifugation is performed under a centrifugal force of 1220g for 5 minutes at a temperature of 20-24 ℃; or centrifuging at 20-24 deg.C under centrifugal force of 700g for 10 min.
9. The method for preparing anticoagulated porcine plasma for thrombus test according to claim 6, wherein the anticoagulated platelet-rich porcine plasma is stored in an environment of 20-24 ℃.
10. Use of anticoagulated platelet-rich porcine plasma obtained by the method for preparing anticoagulated porcine plasma for thrombus test according to claims 6-9 in thrombus test.
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| US20040208786A1 (en) * | 2003-01-27 | 2004-10-21 | Kevy Sherwin V. | Autologous coagulant produced from anticoagulated whole blood |
| CN105092864A (en) * | 2014-05-15 | 2015-11-25 | 北京乐普医疗科技有限责任公司 | 3-level thrombelastogram quality control product and application thereof |
| CN105652022A (en) * | 2015-12-31 | 2016-06-08 | 浙江盛域医疗技术有限公司 | Thromboelastography quality control material and preparation method thereof |
| CN109596845A (en) * | 2018-12-27 | 2019-04-09 | 贵州金玖生物技术有限公司 | A kind of thrombelastogram instrument quality-control product and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208786A1 (en) * | 2003-01-27 | 2004-10-21 | Kevy Sherwin V. | Autologous coagulant produced from anticoagulated whole blood |
| CN105092864A (en) * | 2014-05-15 | 2015-11-25 | 北京乐普医疗科技有限责任公司 | 3-level thrombelastogram quality control product and application thereof |
| CN105652022A (en) * | 2015-12-31 | 2016-06-08 | 浙江盛域医疗技术有限公司 | Thromboelastography quality control material and preparation method thereof |
| CN109596845A (en) * | 2018-12-27 | 2019-04-09 | 贵州金玖生物技术有限公司 | A kind of thrombelastogram instrument quality-control product and preparation method thereof |
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