CN104914254A - Platelet detection method - Google Patents
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Abstract
The present invention relates to the technical field of blood detection, particularly to a platelet detection method, which comprises: mixing a fibrinogen activator and a platelet activator, and dissolving with a buffer to form a mixed reagent; adding an anti-oxidation reagent to the mixed reagent to form an anti-oxidation mixing activation reagent; placing the anti-oxidation mixing activation reagent into a sample cup; placing the sample cup into a penicillin bottle, and carrying out freezing, vacuumizing and capping treatment on the penicillin bottle; taking the sample cup out from the penicillin bottle, placing into a thromboelastogram instrument, and adding the collected whole blood sample into the sample cup; starting the thromboelastogram instrument to detect the whole blood sample mixed with the anti-oxidation mixing activation reagent in the sample cup; and subtracting the fibrinogen strength value from the fibrinogen and platelet composite strength valve so as to obtain the platelet strength value. According to the present invention, the method is simple, the detection result is not interfered by the outside, and the accuracy of the detection result is high.
Description
Technical field
The present invention relates to blood testing technical field, particularly relate to a kind of Platelet Detection.
Background technology
Thrombelastogram was invented in 1948 by German Harter.The 1980's started to be widely used in blood transfusion in clinical guidance art and achieve good result, had now become the most important index of current monitoring coagulation function.Thrombelastogram instrument can export coagulation parameters and the whole processes of fibrinolytic such as blood clotting time R, blood clotting speed Angel and clot strength MA.The physical characteristics of thrombelastogram monitoring blood clot is based on following principle: a special static cylindrical cup filling blood rotated with the angle of 4 ° 45 ', rotate each time and continue 10 seconds.To be hung by taenidium by one and the motion of blood sample monitored by the pin be immersed in blood sample.After cup and pin stick together by fibrin and platelet complex, cup rotates the revolving force produced can be passed to pin in blood sample.The intensity of fibrin and platelet complex can affect the amplitude of needle movement, so that strong blood clot can make the motion of pin and the synchronized movement of cup carry out.Therefore, the motion amplitude of pin and the intensity of established blood clot have direct relation.When blood clot retraction or when dissolving, the connection of pin and blood clot is removed, and the motion of cup no longer passes to pin.The rotation of pin is converted to electronic signal by pickoff, and this electronic signal can be monitored with computer.
At present, fibrinogen and platelet function assay are undertaken by thrombelastogram experiment usually.Wherein, thrombelastogram platelet function assay reagent is made up of common cup detection reagent, fibrin activator, platelet activating agent (as arachidonic acid).The method of existing platelet function assay comprises: when clinical detection, and both are also mixed to form mix reagent by Extemporaneous fibrinogen activator and platelet activating agent; Mix reagent is put into cillin bottle and is prepared into freeze-dried powder; As required quantitative mix reagent distilled water is redissolved; Whole blood sample is added in mix reagent after redissolution; Monitored by the dynamic solidification process of thrombelastogram instrument to whole blood sample, thus quantitatively calculate the fibrinogen of patient and hematoblastic combined strength bination; Record fibrinogenic intensity by thrombelastogram instrument, from fibrinogen and hematoblastic combined strength bination, remove fibrinogenic intensity can obtain hematoblastic intensity.
But, existing by thrombelastogram experiment carry out platelet function assay time, can with weighing in pattern detection process, calculate, redissolve, repeatedly multistep Reagent reconstitution and the reagent pipetting volume process such as application of sample, operation is extremely complicated, methods and results specificity is low before, testing result is often subject to the interference of external operation, its detection accuracy is poor simultaneously, platelet activating agent (as arachidonic acid) is easy to oxidation, platelet activation is caused to lose efficacy, fibrinogen false negative result is there will be in clinical detection, erroneous judgement is caused to clinical, instruct the poor effect of patient medication.
Therefore, for above deficiency, the invention provides a kind of Platelet Detection.
Summary of the invention
(1) technical matters that will solve
The object of this invention is to provide a kind of Platelet Detection is subject to external interference and detection accuracy difference problem with the complicated operation, the testing result that solve existing Platelet Detection and exist.
(2) technical scheme
In order to solve the problems of the technologies described above, the invention provides a kind of Platelet Detection, it comprises the following steps:
S1, by fibrinogen activator and platelet activating agent mixing after by damping fluid carry out dissolvings preparation formation mix reagent;
S2, add in mix reagent anti-oxidant reagent formed anti-oxidant mixing activate reagent;
S3, anti-oxidant mixing is activated reagent put into sample cup;
S4, sample cup is put into cillin bottle, and this cillin bottle is carried out freezing to make the anti-oxidant mixing in sample cup activate reagent freeze-drying and to stick at the bottom of the cup of sample cup;
S5, cillin bottle in step S4 is carried out vacuumizing and gland process, store in vacuum environment to make sample cup;
S6, sample cup to be taken out from cillin bottle, be placed in thrombelastogram instrument, the whole blood sample after gathering is added in sample cup;
S7, startup thrombelastogram instrument detect the whole blood sample being mixed with anti-oxidant mixing activation reagent in sample cup, quantitatively to draw fibrinogen and hematoblastic combined strength bination value.
S8, from fibrinogen and hematoblastic combined strength bination value, remove fibrinogenic intensity level to obtain hematoblastic intensity level.
Wherein, in step S6, whole blood sample to be added in sample cup by sample loading gun and carry out mixed suction, fully mixing with whole blood sample to make anti-oxidant mixing activation reagent.
Wherein, in step S1, described platelet activating agent is arachidonic acid, and the concentration of arachidonic acid in mix reagent is 1 ~ 10mol/L.
Wherein, described buffer reagent comprise in trishydroxymethylaminomethane, phosphate buffer, ethyl sulfonic acid and citric acid one or more, the pH value of described mix reagent is 7.0 ~ 8.0.
Wherein, described fibrinogen activator comprises one or more in fibrin ferment, snake venom thrombin-like enzyme, Batroxobin and Reptilase.
Wherein, in step S1, the concentration of described fibrinogen activator in mix reagent is 0.1 ~ 10U/ml.
Wherein, in step S2, described anti-oxidant reagent comprise in ascorbic acid, arabo-ascorbic acid, vitamin E, lipoic acid and dithiothreitol (DTT) one or more, the concentration that described anti-oxidant reagent activates in reagent in anti-oxidant mixing is 1 ~ 15mol/L.
Wherein, in step s3, sample cup is put into after being mixed into solid-phase reagent in anti-oxidant mixing activation reagent.
Wherein, described solid-phase reagent comprise in sucrose, trehalose, lactose, maltose, glucose and polyglycol one or more, solid-phase reagent is 1%-10% with the anti-oxidant volume ratio scope activating reagent that mixes.
(3) beneficial effect
Technique scheme tool of the present invention has the following advantages: in Platelet Detection provided by the invention, mix reagent is formed by carrying out dissolving preparation by damping fluid after fibrinogen activator and platelet activating agent mixing, then be mixed into anti-oxidant reagent to form anti-oxidant mixing and activate after reagent, add fixating reagent wherein again, obtained reagent is put into sample cup, sample cup puts into cillin bottle, cillin bottle is vacuumized and gland process, store in vacuum environment to make sample cup, in clinical detection process, directly take out the cillin bottle be equipped with in sample cup, open cillin bottle taking-up sample cup namely to test whole blood sample by thrombelastogram instrument, blood platelet test result can be obtained easily, the method is easy and simple to handle, do not need to weigh, calculate, redissolve, the repeatedly troublesome operation step such as application of sample, testing result is not disturbed by external operation, antioxidant effectively can reduce the oxidation rate of platelet activating agent, platelet activating agent is made to be in an inert environments, under sample simultaneously in sample cup is in vacuum environment when storing, improve the stability of reagent in sample cup, and then the accuracy of platelets analysis result can be improved.
Accompanying drawing explanation
Fig. 1 is the structure cut-open view of sample cup and cillin bottle in embodiment of the present invention Platelet Detection.
In figure, 1: cillin bottle; 2: sample cup; 3: bowl cover; 4: sealing-plug; 5: dome; 6: easy open cover.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
In describing the invention, except as otherwise noted, the implication of " multiple " is two or more.Term " on ", D score, " interior ", the orientation of the instruction such as " outward " or position relationship be based on orientation shown in the drawings or position relationship, only the present invention for convenience of description and simplified characterization, instead of indicate or imply that the device of indication or element must have specific orientation, with specific azimuth configuration and operation, therefore can not be interpreted as limitation of the present invention.
Embodiment one
Platelet Detection provided by the invention comprises the following steps:
S1, by fibrinogen activator and platelet activating agent mixing after by damping fluid carry out dissolvings preparation formation mix reagent;
Wherein, the pH value of mix reagent is 7.0-8.0; Fibrinogen activator comprises one or more in fibrin ferment, snake venom thrombin-like enzyme, Batroxobin and Reptilase, and the concentration of fibrinogen activator in mix reagent is 0.1 ~ 10U/ml; Platelet activating agent is arachidonic acid, and the concentration of arachidonic acid in mix reagent is 1 ~ 10mol/L; Buffer reagent comprise in trishydroxymethylaminomethane (Tris), phosphate buffer (PBS), ethyl sulfonic acid and citric acid one or more.
S2, add in mix reagent anti-oxidant reagent formed anti-oxidant mixing activate reagent;
Wherein, anti-oxidant reagent comprise in ascorbic acid, arabo-ascorbic acid, vitamin E, lipoic acid and dithiothreitol (DTT) one or more, the concentration that described anti-oxidant reagent activates in reagent in anti-oxidant mixing is 1 ~ 15mol/L; Antioxidant can effectively reduce arachidonic oxidation rate, and arachidonic acid is in an inert environments.
S3, anti-oxidant mixing is activated reagent put into sample cup; Sample cup comprises glass body and bowl cover, and this sample cup preferably can be placed on thrombelastogram instrument, be convenient to the sample cup of experiment test.
S4, sample cup is put into cillin bottle, and this cillin bottle is carried out freezing to make the anti-oxidant mixing in sample cup activate reagent freeze-drying and to stick at the bottom of the cup of sample cup;
Wherein, cillin bottle is sealed with sealing-plug, is placed on by cillin bottle on freeze dryer pallet, start freeze dryer and carry out freeze-drying, freeze-drying time is 24 hours, wherein can place multiple cillin bottle that sample cup is housed on freeze dryer simultaneously.
S5, cillin bottle in step S4 is carried out vacuumizing and gland process, store in vacuum environment to make sample cup;
Wherein, vacuum tightness is preferably 5 ~ 10Pa.Like this, can ensure that freeze-dried reagent is separated with external environment, avoid reagent to lose efficacy because of the oxygen in ingress of air and moisture, stabilize the performance of reagent, be conducive to the standing storage of reagent.
S6, sample cup to be taken out from cillin bottle, be placed in thrombelastogram instrument, the whole blood sample after gathering is added in sample cup;
S7, startup thrombelastogram instrument detect the whole blood sample being mixed with anti-oxidant mixing activation reagent in sample cup, quantitatively to draw fibrinogen and hematoblastic combined strength bination value.Fibrinogen and hematoblastic combined strength bination value are embodied by fibrinogen and hematoblastic amplitude.
S8, obtain fibrinogenic intensity level according to existing conventional means, from fibrinogen and hematoblastic combined strength bination value, remove fibrinogenic intensity level to obtain hematoblastic intensity level.
When platelets analysis is tested, directly can fetch the cillin bottle that Vacuum Pressure in step S5 is built, open cillin bottle, take out sample cup wherein, sample cup is placed on thrombelastogram instrument, then the whole blood of patient is gathered, the whole blood sample gathered is injected sample cup, carry out detection and can obtain fibrinogen and hematoblastic combined strength bination value, then remove and obtain fibrinogenic intensity level by conventional means, namely can obtain hematoblastic intensity level, realize hematoblastic detection.
In actual applications, hematoblastic function value is detected by method of the present invention after patient takes antiplatelet drug aspirin, then the fibrinogen function value of patient is cut, just can obtain the platelet function net value after patient's Aspirin, by contrasting with patient's blood platelet net value of not taking medicine, can show that patient's blood platelet after the tablet has been ingested suppresses ratio, thus reflection patient antiplatelet drug therapeutic effect of aspirin indirectly, instruct cardiovascular patient clinical treatment and more after.
By method of the present invention in clinical detection process without the need to adding activation reagent, without the need to carrying out the preprocessing process of blood preparation, can direct-detection after blood specimen collection.Thrombelastogram instrument meeting automatic analysis detects blood platelet amplitude, fibrin amplitude, the fibrin ferment amplitude of patient whole blood's sample, the antiplatelet drug inhibiting rate of patient can be drawn by particular analysis formula, quantitative test Patient drug action effect, instruct fast cardio-cerebral vascular disease patient clinical diagnosis, treatment and more after, be clinically provide necessary drug effect monitoring foundation.
Further, in step s 6, whole blood sample to be added in sample cup by sample loading gun and carry out mixed suction, fully mixing with whole blood sample to make anti-oxidant mixing activation reagent.Wherein, the mixed number of times inhaled is generally three times.
Preferably, in step s3, sample cup is put into after being mixed into solid-phase reagent in anti-oxidant mixing activation reagent;
Wherein, described solid-phase reagent comprise in sucrose, trehalose, lactose, maltose, glucose and polyglycol (PEG) one or more, solid-phase reagent is 1%-10% with the anti-oxidant volume ratio scope activating reagent that mixes.Solid-phase reagent can fully be combined with sample cup bottom even, and this solid-phase reagent has good diffusion effect after mixing with blood, dissolves in blood fast, ensure that the stability of blood examination after sample adds.
Embodiment two
As shown in Figure 1, on the basis of embodiment one, embodiment two discloses a kind of sample cup 2 and cillin bottle 1 of Platelet Detection.
Particularly, sample cup 2 comprises glass body and bowl cover 3, and bowl cover 3 main body is hollow circular cylinder, and cylinder top end has the edge that circumference extends, and bowl cover 3 main body extend in glass chamber; Sample cup 2 is contained in cillin bottle 1, the bottleneck of cillin bottle 1 is provided with sealing-plug 4, the lower end of sealing-plug 4 is fitted on the bowl cover 3 of sample cup, easy open cover 6 is equipped with in cillin bottle 1 upper end, dome 5 is provided with between easy open cover 6 and sealing-plug 4, cavity in the bowl cover 3 of sample cup is used for fixing metal syringe needle, coordinates the use of thrombelastogram instrument; The lower end of sealing-plug 4 is fitted on the bowl cover 3 of sample cup, in the process that sample cup 2 and cillin bottle 1 store and shift, prevent sample cup 2 from moving.
During use, first sample cup 2 is positioned in cillin bottle 1; Secondly, fibrinogen is activated reagent, platelet activation reagent and damping fluid and quantitatively join in the cavity in sample cup 2, and the bowl cover 3 of sample cup is contained in it the cup of sample cup; Then, sample cup 2 and cillin bottle 1 are positioned on freeze dryer carry out freeze-drying; Finally, press sealing-plug 4 under vacuum and encapsulate, then load onto dome 5 and easy open cover 6.
In sum, in Platelet Detection provided by the invention, mix reagent is formed by carrying out dissolving preparation by damping fluid after fibrinogen activator and arachidonic acid mixing, then be mixed into anti-oxidant reagent to form anti-oxidant mixing and activate after reagent, add fixating reagent wherein again, obtained reagent is put into sample cup, and sample cup puts into cillin bottle, cillin bottle is vacuumized and gland process, store in vacuum environment to make sample cup; In clinical detection process, directly take out the cillin bottle be equipped with in sample cup, open cillin bottle taking-up sample cup namely to test whole blood sample by thrombelastogram instrument, obtain blood platelet test result easily, the method is easy and simple to handle, do not need the troublesome operation step such as weighing, calculating, redissolution, repeatedly application of sample, testing result is not disturbed by external operation, antioxidant can effectively reduce arachidonic oxidation rate, make arachidonic acid be in an inert environments, improve detection accuracy; Solid-phase reagent wherein can fully be combined with sample cup bottom even, and this solid-phase reagent has good diffusion effect after mixing with blood, dissolves in blood fast, ensure that the stability of blood examination after blood sample adds.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (9)
1. a Platelet Detection, is characterized in that: it comprises the following steps:
S1, by fibrinogen activator and platelet activating agent mixing after by damping fluid carry out dissolvings preparation formation mix reagent;
S2, add in mix reagent anti-oxidant reagent formed anti-oxidant mixing activate reagent;
S3, anti-oxidant mixing is activated reagent put into sample cup;
S4, sample cup is put into cillin bottle, and this cillin bottle is carried out freezing to make the anti-oxidant mixing in sample cup activate reagent freeze-drying and to stick at the bottom of the cup of sample cup;
S5, cillin bottle in step S4 is carried out vacuumizing and gland process, store in vacuum environment to make sample cup;
S6, sample cup to be taken out from cillin bottle, be placed in thrombelastogram instrument, the whole blood sample after gathering is added in sample cup;
S7, startup thrombelastogram instrument detect the whole blood sample being mixed with anti-oxidant mixing activation reagent in sample cup, quantitatively to draw fibrinogen and hematoblastic combined strength bination value.
S8, from fibrinogen and hematoblastic combined strength bination value, remove fibrinogenic intensity level to obtain hematoblastic intensity level.
2. Platelet Detection according to claim 1, is characterized in that: in step S6, to be added by whole blood sample in sample cup and carries out mixed suction, fully mix to make anti-oxidant mixing activation reagent with whole blood sample by sample loading gun.
3. Platelet Detection according to claim 1, is characterized in that: in step S1, and described platelet activating agent is arachidonic acid, and the concentration of arachidonic acid in mix reagent is 1 ~ 10mol/L.
4. Platelet Detection according to claim 1, is characterized in that: described buffer reagent comprise in trishydroxymethylaminomethane, phosphate buffer, ethyl sulfonic acid and citric acid one or more, the pH value of described mix reagent is 7.0 ~ 8.0.
5. Platelet Detection according to claim 1, is characterized in that: described fibrinogen activator comprises one or more in fibrin ferment, snake venom thrombin-like enzyme, Batroxobin and Reptilase.
6. Platelet Detection according to claim 1, is characterized in that: in step S1, and the concentration of described fibrinogen activator in mix reagent is 0.1 ~ 10U/ml.
7. Platelet Detection according to claim 1, it is characterized in that: in step S2, described anti-oxidant reagent comprise in ascorbic acid, arabo-ascorbic acid, vitamin E, lipoic acid and dithiothreitol (DTT) one or more, the concentration that described anti-oxidant reagent activates in reagent in anti-oxidant mixing is 1 ~ 15mol/L.
8. Platelet Detection according to claim 1, is characterized in that: in step s3, puts into sample cup after being mixed into solid-phase reagent in anti-oxidant mixing activation reagent.
9. Platelet Detection according to claim 8, it is characterized in that: described solid-phase reagent comprise in sucrose, trehalose, lactose, maltose, glucose and polyglycol one or more, solid-phase reagent is 1%-10% with the anti-oxidant volume ratio scope activating reagent that mixes.
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| CN109884326A (en) * | 2019-03-27 | 2019-06-14 | 深圳优迪生物技术有限公司 | Platelet aggregation detection kit |
| CN110514823A (en) * | 2019-08-28 | 2019-11-29 | 深圳麦科田生物医疗技术有限公司 | A kind of blood platelet inhibiting rate detection kit and preparation method thereof and detection method |
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| CN109884326A (en) * | 2019-03-27 | 2019-06-14 | 深圳优迪生物技术有限公司 | Platelet aggregation detection kit |
| CN109884326B (en) * | 2019-03-27 | 2022-08-19 | 深圳优迪生物技术有限公司 | Platelet aggregation function detection kit |
| CN110514823A (en) * | 2019-08-28 | 2019-11-29 | 深圳麦科田生物医疗技术有限公司 | A kind of blood platelet inhibiting rate detection kit and preparation method thereof and detection method |
| CN110514823B (en) * | 2019-08-28 | 2023-12-05 | 深圳麦科田生物医疗技术股份有限公司 | Platelet inhibition rate detection kit, preparation method and detection method thereof |
| CN112730769A (en) * | 2021-01-29 | 2021-04-30 | 郑州普湾医疗技术有限公司 | Platelet aggregation functional adenosine diphosphate cup detection reagent and preparation method thereof |
| CN112924701A (en) * | 2021-01-29 | 2021-06-08 | 郑州普湾医疗技术有限公司 | Batroxobin cup detection reagent with platelet aggregation function and preparation method thereof |
| CN113156144A (en) * | 2021-01-29 | 2021-07-23 | 郑州普湾医疗技术有限公司 | Arachidonic acid cup detection reagent with platelet aggregation function and preparation method thereof |
| CN113848332A (en) * | 2021-09-17 | 2021-12-28 | 广州徕西姆医学诊断技术有限公司 | Thrombus elastogram detection reagent and preparation method and application thereof |
| CN113848332B (en) * | 2021-09-17 | 2024-04-19 | 广州徕西姆医学诊断技术有限公司 | Thrombus elastography detection reagent and preparation method and application thereof |
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Address after: 102200, Beijing Changping District science and Technology Park, super Road, No. 7-1, building 37 Patentee after: Beijing Lepu Diagnostic Technology Co., Ltd Address before: 102200, Beijing Changping District science and Technology Park, super Road, No. 7-1, building 37 Patentee before: BEIJING LEPU MEDICAL TECHNOLOGY Co.,Ltd. |