CN105445478B - APTT kit and preparation method thereof - Google Patents
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Abstract
The invention discloses a kind of APTT kit and preparation method thereof, the APTT kit eliminates packing in process for preparation and the step such as lyophilized, avoid because of difference between the bottle of the reagent caused by lyophilized and redissolution process, so that the larger difference between causing measurement result.Reagent of the present invention is without lyophilized, you can ensure the stability of reagent, it is ensured that the optimal validity of reagent continues at least one moon after corkage, avoid the larger difference between experimental result, the consumption of reagent has been saved simultaneously, and corkage is used, operated more fast and simple.
Description
Technical field
The present invention relates to a kind of external diagnosis reagent case and preparation method thereof, more particularly to a kind of activated partial APTT
Timing kit and preparation method thereof, belongs to biological technical field.
Background technology
The hemostatic function of body is by the collective effect of blood platelet, blood coagulation system, fibrinolytic system and blood vessel endothelium system etc.
Come what is completed.Past we once with Screen test of the bleeding time as hemostatic function defect, but because of its operational standardization degree
Not high, sensitiveness is bad, and can not reflect the content and activity of clotting factor, therefore is replaced by coagulation function inspection at present.
Coagulation function inspection is mainly blood coagulation four --- prothrombin time, activated partial thromboplastin time, thrombin time and
Fibrinogen content.Blood coagulation four is the important visiting project of clinical laboratory, is also one of project that operation consent must be detected, is coagulated
Four detections of blood are significant to the coagulation function for understanding check object.
It is that the intrinsic coagulation factor lacks most reliable screening test that activated partial thromboplastin time (APTT), which is determined, is
For confirming congenital or acquired platelet cofactor Ⅰ, Ⅸ, Ⅺ defect or with the presence or absence of their corresponding mortifiers.APTT prolongs
Length is seen：(1) blood plasma level of I2GdBN, Ⅸ and Ⅺ lowers, such as hemophilia.(2) serious factor (factor II) Factor Ⅴ,
Ⅹ and fibrinogenopenia, such as liver diseases, obstructive jaundice, hemorrhagic disease of newborn, enteron aisle sterilizing syndrome, malabsorption
Syndrome, oral anticoagulant, using heparin and low (nothing) fibrinogenemia.(3) fine appearance vigor enhancing, such as Secondary cases or
There is fibrin (original) degradation product (FDP) in primary fibrinolytic and blood circulation.(4) have anticoagulant substances in blood circulation, such as it is anti-because
Sub VIII or Ⅸ antibody, SLE etc..APTT, which shortens, to be seen：(1) the high blood coagulation phase of hypercoagulative state, such as DIC, coagulant enters blood flow
And the activity of clotting factor increases.(2) thrombotic diseases, such as miocardial infarction, unstable angina, cerebrovascular disease,
Diabetes mellitus vascular lesion, pulmonary infarction, Deep vain thrombosis, pregnancy-induced hypertension syndrome and nephrotic syndrome etc..
Because commercially available activated partial thromboplastin time (APTT) is determined, kit species is various and quality is uneven,
So that larger with the result difference that a patient determines in different laboratories so that testing result is inaccurate, so as to influence doctor
The raw correct Clinics and Practices to disease.Therefore, activated partial thromboplastin time (APTT) determines the quality of reagent into standard
True measurement result and the key factor of correct diagnoses and treatment.Simultaneously as many powdered reagents are used after need to redissolving, and answer
Reagent stability after molten is again poor, can be made troubles again to the APTT operations determined and cause unnecessary waste, therefore,
Develop a kind of effective activated partial thromboplastin time (APTT) measure reagent reliable, easy to use undoubtedly reasonably necessary
's.
The content of the invention
In order to overcome the shortcomings of in above-mentioned existing background technology, the present invention provides a kind of activated partial thromboplastin time and surveyed
Determine kit and preparation method thereof, the APTT kit eliminate packing in process for preparation and
The step such as lyophilized, it is to avoid reagent stability caused by being redissolved during use and determine the problem of effect is deteriorated, it is ensured that examination
The stability of agent, while it also avoid the waste of reagent, further obtains the overall performances such as accuracy, repeatability and stability more
The good and easier APTT reagent of operation.
To achieve these goals, the present invention provides a kind of APTT kit, includes phosphorus
Fat reagent and chlorination calcon, two kinds of reagents are stored in different vessels；
The phospholipid reagent is mixed by solid activator, phosphatide and buffer system, the content of the solid activator
For 0.01~2g/L, the solid activator is selected from ellagic acid, bentonite, kaolin, diatomite, aluminium-magnesium silicate one of which or several
The mixture planted；The content of the phosphatide is 2.5~8wt%, and the pH of the buffer system is 6.0~8.0, the buffer system
Middle each component content is to include：0.1~1M buffer, 0.01~0.2wt% stabilizer, 0.1~1.0wt% poly- second two
Alcohol, remaining be water；The buffer is selected from：Tris-Hcl、Hepes、PBS、MPOS；The stabilizer is selected from：Sodium azide,
Kathon, Proclin, glycine, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid；
The working concentration of the chlorination calcon is 15~40mM.
Preferably, in the above-mentioned technical solutions, the content of the solid activator is 0.05~1g/L.
Preferably, in the above-mentioned technical solutions, the content of phospholipid is 5~7wt%.
Preferably, in the above-mentioned technical solutions, the phosphatide is rabbit cephalin.
Preferably, in the above-mentioned technical solutions, content of the buffer in buffer system is 0.4~0.75M.
Preferably, in the above-mentioned technical solutions, content of the stabilizer in buffer system is 0.05~0.1wt%,
It is highly preferred that the stabilizer is Kathon, glycine, sero-abluminous 1:0.86:1.1 mixture.
Preferably, in the above-mentioned technical solutions, the stabilizer is selected from：Sodium azide, Kathon, Proclin, sweet ammonia
Acid, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid.
Preferably, in the above-mentioned technical solutions, content of the polyethylene glycol in buffer system is 0.3~0.8wt%.
Preferably, in the above-mentioned technical solutions, the working concentration of the chlorination calcon is 20~35mM.
The present invention also provides a kind of method for preparing APTT kit described above, including
Following steps：(1) prepares buffer system first, and pH is adjusted to 6.0~8.0, adds after solid activator and mixes, obtains initial mixing
Solution；(2) phosphatide that various concentrations are added into initial mixing solution is mixed, and coordinates calcium chloride solution to carry out matter on Blood coagulation instrument
Control determine, measurement result closest to the ratio of reagents of the Quality Control scope median of the type Blood coagulation instrument be exactly final proportioning；(3)
Matched according to final reagent, phosphatide is added into initial mixing solution, polyethylene glycol and stabilizer are added after stirring and evenly mixing, mixed
It is the phospholipid reagent in APTT kit after even；(4) calcium chloride solid dissolving is weighed in one
In level water, the chlorination calcon being in APTT kit is mixed；(5) by phospholipid reagent
APTT kit is obtained after being packed respectively with chlorination calcon.
Beneficial effects of the present invention：A kind of APTT kit and preparation method thereof is provided,
The APTT kit eliminates packing in process for preparation and the step such as lyophilized, it is to avoid because freezing
Difference between the bottle for the reagent that dry and redissolution process is caused, so that the larger difference between causing measurement result.Reagent of the present invention without
It need to freeze, you can ensure the stability of reagent, it is ensured that the optimal validity of reagent continues at least one moon after corkage, it is to avoid
Larger difference between experimental result, while having saved the consumption of reagent, corkage is used, and is operated more fast and simple.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
The APTT kit of the present invention, includes phospholipid reagent and chlorination calcon, two kinds
Reagent is stored in different containers；
Phospholipid reagent is made up of solid activator, phosphatide and buffer system, and the content of solid activator is 0.01~2g/L,
The content of phosphatide is 2.5~8wt%, and the pH of buffer system is that each component content is in 6.0~8.0, buffer system：0.1~1M
Buffer, 0.01~0.2wt% stabilizer, 0.1~1.0wt% polyethylene glycol, remaining be water；The buffer is selected from
In：Tris-Hcl、Hepes、PBS、MPOS；The stabilizer is selected from：Sodium azide, Kathon, Proclin, glycine, ox blood
Pure albumen, thimerosal, mercuric nitrate benzene, chloroform, carbolic acid；
The working concentration of chlorination calcon is 15~40mM.
The method for preparing above-mentioned APTT kit, comprises the following steps：(1) buffering is prepared
System, pH is adjusted to 6.0~8.0, adds after solid activator and mixes, obtains initial mixing solution；(2) into initial mixing solution
The phosphatide for adding various concentrations is mixed, and coordinates calcium chloride solution to carry out Quality Control measure on Blood coagulation instrument, measurement result is closest should
The ratio of reagents of the Quality Control scope median of type Blood coagulation instrument is exactly final proportioning；(3) matched according to final reagent, to first
Phosphatide is added in beginning mixed solution, polyethylene glycol and stabilizer are added after stirring and evenly mixing, is that activated partial blood coagulation is lived after mixing
Phospholipid reagent in enzyme timing kit；(4) calcium chloride solid dissolving is weighed in one-level water, is mixed and is activation
Partial thromboplastin time determines the chlorination calcon in kit；(5) after phospholipid reagent and chlorination calcon being packed respectively
Obtain APTT kit.
Embodiment 1
APTT kit, includes phospholipid reagent and chlorination calcon, two kinds of reagent storages
In different vessels；Phospholipid reagent is made up of ellagic acid, phosphatide and buffer system, the content of ellagic acid is 2g/L, and phosphatide contains
Measure as 2.5wt%, each component content is in buffer system：1M Tris-Hcl, 0.01wt% Kathon, 1.0wt%'s is poly-
Ethylene glycol, remaining be water, the pH of buffer system is 6.0；The working concentration of chlorination calcon is 15mM.Use kit of the present invention
When, the volume ratio of test plasma and phospholipid reagent consumption is 1:0.85~1.1.
The method for preparing above-mentioned APTT kit, comprises the following steps：(1) buffering is prepared
System, pH is adjusted to 6.0~8.0, adds after solid activator and mixes, obtains initial mixing solution；(2) into initial mixing solution
The phosphatide for adding various concentrations is mixed, and coordinates calcium chloride solution to carry out Quality Control measure, measurement result most type on Blood coagulation instrument
The ratio of reagents of the Quality Control scope median of Blood coagulation instrument is exactly final proportioning；(3) match, mixed to initial according to final reagent
Close in solution and add phosphatide, polyethylene glycol and stabilizer are added after stirring and evenly mixing, when being activated partial APTT after mixing
Between determine kit in phospholipid reagent；(4) calcium chloride solid dissolving is weighed in one-level water, is mixed and is activated partial
APFI determines the chlorination calcon in kit；(5) obtained after packing phospholipid reagent and chlorination calcon respectively
APTT kit.
The kit obtained using the present embodiment determines the prothrombin time of test plasma by semi-automatic coagulo meter, surveys
The method of determining is：First phospholipid reagent is balanced to room temperature, chlorination calcon warm bath is pressed to 37 DEG C, then by test plasma with phospholipid reagent
It is 1 according to volume ratio:0.85~1.1 ratio is mixed, and is placed in 37 DEG C of warm bath 3min, is added chlorination calcon, makes its final concentration
For 15~40mM, timing is mixed at once, records prothrombin time.Ten parallel laboratory tests are done, record data analyzes the present embodiment
Reagent repeatability such as table 1 in the repeatability of reagent, the present embodiment.
Embodiment 2
APTT kit, includes phospholipid reagent and chlorination calcon, two kinds of reagent storages
In different vessels；The phospholipid reagent is made up of bentonite, rabbit cephalin and buffer system, and the content of bentonite is 0.05g/L, rabbit
The content of cephalin is that the content of each component in 7wt%, buffer system is：0.4M MPOS, 0.1wt% Sodium azide,
0.8wt% polyethylene glycol, remaining be water；The pH of buffer system is 7.8, and the working concentration of chlorination calcon is 20mM.
APTT reagent box preparation method be the same as Example 1.
The kit obtained using the present embodiment determines the prothrombin time of test plasma on semi-automatic coagulo meter, surveys
Determine method be the same as Example 1, do ten parallel laboratory tests, record data analyzes the repeatability of the present embodiment reagent, the present embodiment pilot scale
Agent repeatability such as table 1.
Embodiment 3
APTT kit, includes phospholipid reagent and chlorination calcon, two kinds of reagent storages
In different vessels；Phospholipid reagent is made up of solid activator, rabbit cephalin and buffer system, and the content of solid activator is
1.0g/L, the content of rabbit cephalin is that the content of each component in 5wt%, buffer system is：0.75M Hepes, 0.05wt%'s
Stabilizer, 0.3wt% polyethylene glycol, remaining be water；The pH of buffer system is 6.0, and the working concentration of chlorination calcon is
35mM；Wherein, solid activator is that ellagic acid, kaolin and aluminium-magnesium silicate quality are 1:0.5:2 mixture, stabilizer is
Kathon, glycine, sero-abluminous 1:0.86:1.1 mixture.
APTT reagent box preparation method be the same as Example 1.
The kit obtained using the present embodiment determines the prothrombin time of test plasma on semi-automatic coagulo meter, surveys
Determine method be the same as Example 1, do ten parallel laboratory tests, record data analyzes the repeatability of the present embodiment reagent, the present embodiment pilot scale
Agent repeatability such as table 1.
Embodiment 4
APTT kit, includes phospholipid reagent and chlorination calcon, two kinds of reagent storages
In different vessels；Phospholipid reagent is made up of diatomite, rabbit cephalin and buffer system, and diatomaceous content is 1.5g/L, rabbit
The content of cephalin is that the content of each component in 3wt%, buffer system is：0.25M PBS, 0.03wt% Proclin,
0.9wt% polyethylene glycol, remaining be water；The pH of buffer system is 7.0, and the working concentration of chlorination calcon is 18mM.
APTT reagent box preparation method be the same as Example 1.
The kit obtained using the present embodiment determines the prothrombin time of test plasma on semi-automatic coagulo meter, surveys
Determine method be the same as Example 1, do ten parallel laboratory tests, record data analyzes the repeatability of the present embodiment reagent, the present embodiment pilot scale
Agent repeatability such as table 1.
Embodiment 5
APTT kit, includes phospholipid reagent and chlorination calcon, two kinds of reagent storages
In different vessels；Phospholipid reagent is made up of aluminium-magnesium silicate, rabbit cephalin and buffer system, and the content of aluminium-magnesium silicate is 1.5g/
L, the content of rabbit cephalin is that the content of each component in 8wt%, buffer system is：0.1M Tris-Hcl, 0.2wt% coal
Acid, 0.1wt% polyethylene glycol, remaining be water；The pH of buffer system is 8.0, and the working concentration of chlorination calcon is 40mM.
APTT reagent box preparation method be the same as Example 1.
The kit obtained using the present embodiment determines the prothrombin time of test plasma on semi-automatic coagulo meter, surveys
Determine method be the same as Example 1, do ten parallel laboratory tests, record data analyzes the repeatability of the present embodiment reagent, the present embodiment pilot scale
Agent repeatability such as table 1.
Experimental example
Experimental example 1
The kit that Application Example 1~5 is obtained determines the prothrombin time of test plasma by semi-automatic coagulo meter,
Assay method is：First phospholipid reagent is balanced to room temperature, chlorination calcon warm bath is to 37 DEG C, then by test plasma and phospholipid reagent
It is 1 according to volume ratio:1 ratio is mixed, and is placed in 37 DEG C of warm bath 3min, is added chlorination calcon, makes its final concentration of 25mM,
Timing is mixed at once, records prothrombin time.The kit of each embodiment does ten parallel laboratory tests, record data.Use city
Sell reagent and determine same plasma sample, record prothrombin time, reagent of the present invention is compared knot with commercial reagent repeatability
Fruit such as table 1.
Table 1：Reagent of the present invention is compared with commercial reagent repeatability
Table 1 shows that reagent of the present invention is compared with commercial reagent, and the coefficient of variation (CV) value is smaller, reproducible
Experimental example 2
Compare the stability of reagent of the present invention and commercial reagent, reagent of the present invention and commercial reagent are opened simultaneously, it is commercially available
Reagent is to be used after freeze-dried powder redissolves, and reagent of the present invention is directly used, and does Quality Control test on identical Blood coagulation instrument, and record is with opening
The change reagent of the present invention and the Quality Control result of commercial reagent on date after bottle, the two stability compare such as table 2.
Table 2：Reagent of the present invention is compared with commercial reagent stability
| Reagent | 1 day | 3 days | 7 days | 15 days | 20 days | 30 days | 40 days |
| Embodiment 3 | 32.0 | 31.8 | 31.7 | 31.8 | 31.8 | 32.7 | 32.9 |
| Commercial reagent | 34.7 | 33.6 | 36.2 | 37.3 | 38.9 | 40.2 | 42.6 |
Table 2 shows that the prothrombin time value of reagent of the present invention had no significant change in 40 days, and commercial reagent is solidifying
Hemase original time value was just changed from the 7th day, and with the increase of number of days, its prothrombin time value becomes big in obvious
Trend, illustrates that the stability of reagent of the present invention is preferable.
Claims (2)
1. a kind of APTT kit, it is characterised in that：Comprising phospholipid reagent and chlorination calcon,
Two kinds of reagents are stored in different vessels；
The phospholipid reagent is made up of solid activator, rabbit cephalin and buffer system；
The content of the solid activator is 1.0g/L, is that ellagic acid, kaolin and aluminium-magnesium silicate quality are 1:0.5:2 mixing
Thing；
The content of the rabbit cephalin is 5wt%；
The content of each component is in the buffer system：0.75M Hepes, 0.05wt% stabilizer, 0.3wt% poly- second
Glycol, remaining be water；The pH of buffer system is 6.0, wherein, stabilizer is Kathon, glycine, sero-abluminous 1:
0.86:1.1 mixture；
The working concentration of the chlorination calcon is 35mM.
2. prepare the method for APTT kit described in claim 1, it is characterised in that：Including such as
Lower step：(1) buffer system is prepared, pH is adjusted to 6.0~8.0, adds after solid activator and mix, obtain initial mixing solution；
(2) rabbit cephalin that various concentrations are added into initial mixing solution is mixed, and coordinates calcium chloride solution to carry out matter on Blood coagulation instrument
Control determine, measurement result closest to the ratio of reagents of the Quality Control scope median of the type Blood coagulation instrument be exactly final proportioning；(3)
Matched according to final reagent, rabbit cephalin is added into initial mixing solution, polyethylene glycol and stably is added after stirring and evenly mixing
Agent, is the phospholipid reagent in APTT kit after mixing；(4) calcium chloride solid dissolving is weighed
In one-level water, the chlorination calcon being in APTT kit is mixed；(5) by phosphatide
Reagent and chlorination calcon obtain APTT kit after packing respectively.
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105353141B (en) * | 2015-11-17 | 2018-04-10 | 北京美创新跃医疗器械有限公司 | Detection reagent and its application and the kit containing the reagent |
| CN106290923B (en) * | 2016-08-01 | 2018-05-11 | 重庆鼎润医疗器械有限责任公司 | One kind activation coagulation assay reagent and its application |
| CN107942080A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of activated partial thromboplastin time detection reagent and its detection method |
| CN107445972A (en) * | 2017-07-27 | 2017-12-08 | 陕西科技大学 | A kind of biodegradable PBS bases aliphatic polyester nucleator and preparation method thereof |
| CN107748267B (en) * | 2017-10-13 | 2019-02-26 | 山东艾科达生物科技有限公司 | A kind of kit measuring activated partial thromboplastin time (APTT) |
| CN108169152A (en) * | 2017-12-27 | 2018-06-15 | 山东博科生物产业有限公司 | A kind of angiotensin converting enzyme detection kit and its application method |
| CN108279313B (en) * | 2017-12-29 | 2021-03-02 | 广州万孚生物技术股份有限公司 | Reagent and test cup for rapidly detecting blood viscoelasticity |
| CN110108890A (en) * | 2019-05-05 | 2019-08-09 | 深圳优迪生物技术有限公司 | Activated partial thromboplastin time assay reagent and its application |
| CN110412001B (en) * | 2019-08-01 | 2022-02-11 | 武汉塞力斯生物技术有限公司 | Kit for dynamically monitoring thrombin generation capacity |
| CN110824154A (en) * | 2019-10-15 | 2020-02-21 | 常熟常江生物技术有限公司 | Activator for thromboelastography |
| CN111638375B (en) * | 2020-06-08 | 2022-12-13 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for measuring activated partial thromboplastin time |
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| US5506146A (en) * | 1993-06-30 | 1996-04-09 | Stiftung Fur Diagnostische Forschung | Measurement of the activated partial thromboplastin time (APTT) in a one-step reaction |
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| CN101226201B (en) * | 2008-02-05 | 2011-05-11 | 长安大学 | Agent for activation sector cruor activating-enzyme time (APTT) |
| JP2009243955A (en) * | 2008-03-28 | 2009-10-22 | Sekisui Chem Co Ltd | Method for measuring concentration of marker related to blood coagulation |
| DE102009040880B4 (en) * | 2009-09-09 | 2012-10-18 | Andreas Hettich Gmbh & Co. Kg | Apparatus for performing hemostasis tests |
| WO2011049431A1 (en) * | 2009-10-19 | 2011-04-28 | Stichting Katholieke Universiteit, Radboud Universiteit Nijmegen | Blood coagulation status measurement in saliva |
| JP2014190954A (en) * | 2013-03-28 | 2014-10-06 | Sysmex Corp | Reagent for activated partial thromboplastin time measurement and usage thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5506146A (en) * | 1993-06-30 | 1996-04-09 | Stiftung Fur Diagnostische Forschung | Measurement of the activated partial thromboplastin time (APTT) in a one-step reaction |
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