CN105445478A - Activated partial thromboplastin time measurement kit and preparation method thereof - Google Patents
Activated partial thromboplastin time measurement kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an activated partial thromboplastin time measurement kit and a preparation method thereof. By the adoption of the activated partial thromboplastin time measurement kit, the steps of subpackaging, freeze-drying and the like in the preparation process are eliminated, and the condition that a greater difference between measurement results is caused due to between-bottle difference caused by freeze-drying and redissolving processes of reagents is avoided. The reagents used in the activated partial thromboplastin time measurement kit do not need to be freeze-dried, to guarantee the stability of the reagents and ensure that the optimal effectiveness of the decapped reagents lasts for at least one month, thus avoiding the greater difference between experimental results; meanwhile, the use amount of the reagents is reduced; the reagents can be used instantly after a bottle is opened, and the operation is quicker, simpler and more convenient.
Description
Technical field
The present invention relates to a kind of external diagnosis reagent case and preparation method thereof, particularly relate to a kind of APTT kit and preparation method thereof, belong to biological technical field.
Background technology
The hemostatic function of body has been come by the acting in conjunction of blood platelet, blood coagulation system, fibrinolytic system and blood vessel endothelium system etc.Past we once used the bleeding time as the Screen test of hemostatic function defect, but because of its operational standardization degree not high, susceptibility is bad, and cannot reflect content and the activity of clotting factor, therefore at present by coagulation function inspection is replaced.Coagulation function checks mainly blood coagulation four---prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen content.The important visiting project of blood coagulation four Xiang Shi clinical laboratory, be also one of operation consent project that must detect, the coagulation function that blood coagulation four detects understanding check object is significant.
It is that the intrinsic coagulation factor lacks the most reliable shaker test that activated partial thromboplastin time (APTT) measures, be used to confirm congenital or acquired platelet cofactor Ⅰ, Ⅸ, the defect of Ⅺ or whether there is their corresponding mortifiers.APTT prolongation sees: (1) I2GdBN, Ⅸ and Ⅺ blood plasma level lower, as hemophilia.(2) serious factor (factor II) Factor Ⅴ, Ⅹ and fibrinogenopenia, as liver diseases, obstructive jaundice, hemorrhagic disease of newborn, enteron aisle sterilizing syndrome, malabsorption syndrome, oral anticoagulant, application heparin and low (nothing) fibrinogenemia.(3) fine appearance vigor strengthens, as having fibrin (former) degradation product (FDP) in Secondary cases or primary fibrinolytic and blood circulation.(4) anticoagulant substances is had in blood circulation, as anti-I2GdBN or Ⅸ antibody, SLE etc.APTT shortening sees: (1) hypercoagulative state, as the high blood coagulation phase of DIC, the activity that coagulant enters blood flow and clotting factor increases.(2) thrombotic diseases, 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) measures that kit is of a great variety and quality is uneven, the result difference that a same patient is measured in different laboratories is larger, make testing result inaccurate, thus affect the correct Clinics and Practices of doctor to disease.Therefore, the quality that activated partial thromboplastin time (APTT) measures reagent has become the key factor of measurement result and correct diagnoses and treatment accurately.Simultaneously, because a lot of powdered reagent need redissolve rear use, and redissolve after reagent stability poor, the operation that can measure to APTT again makes troubles and causes unnecessary waste, therefore, developing a kind of activated partial thromboplastin time effectively reliable, easy to use (APTT), to measure reagent beyond doubt quite necessary.
Summary of the invention
In order to overcome the deficiency in above-mentioned existing background technology, the invention provides a kind of APTT kit and preparation method thereof, this APTT kit eliminates the step such as packing and freeze-drying in process for preparation, avoid in use procedure redissolve institute cause reagent stability and mensuration deleterious problem, ensure that the stability of reagent, it also avoid the waste of reagent simultaneously, obtain accuracy further, repeatability and the better and APTT reagent that operation is easier of the overall performance such as stability.
To achieve these goals, the invention provides a kind of APTT kit, comprise phospholipid reagent and lime chloride reagent, two kinds of reagent are stored in different vessels;
Described phospholipid reagent is mixed by solid activator, phosphatide and buffer system, the content of described solid activator is 0.01 ~ 2g/L, and described solid activator is selected from ellagic acid, bentonite, porcelain earth, zeyssatite, aluminium-magnesium silicate wherein one or more potpourri; The content of described phosphatide is 2.5 ~ 8wt%, the pH of described buffer system is 6.0 ~ 8.0, and in described buffer system, each component concentration is comprise: the polyglycol of the buffering agent of 0.1 ~ 1M, the stabilizing agent of 0.01 ~ 0.2wt%, 0.1 ~ 1.0wt%, all the other are water; Described buffering agent is selected from: Tris-Hcl, Hepes, PBS, MPOS; Described stabilizing agent is selected from: Sodium azide, Kathon, Proclin, glycocoll, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid;
The working concentration of described lime chloride reagent is 15 ~ 40mM.
Preferably, in technique scheme, the content of described solid activator is 0.05 ~ 1g/L.
Preferably, in technique scheme, described content of phospholipid is 5 ~ 7wt%.
Preferably, in technique scheme, described phosphatide is rabbit cephalin.
Preferably, in technique scheme, the content of described buffering agent in buffer system is 0.4 ~ 0.75M.
Preferably, in technique scheme, the content of described stabilizing agent in buffer system is 0.05 ~ 0.1wt%, and more preferably, described stabilizing agent is the potpourri of Kathon, glycocoll, sero-abluminous 1:0.86:1.1.
Preferably, in technique scheme, described stabilizing agent is selected from: Sodium azide, Kathon, Proclin, glycocoll, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid.
Preferably, in technique scheme, the content of described polyglycol in buffer system is 0.3 ~ 0.8wt%.
Preferably, in technique scheme, the working concentration of described lime chloride reagent is 20 ~ 35mM.
The present invention also provides a kind of method preparing APTT kit described above, comprise the steps: first (1) preparation buffer system, pH is adjusted to 6.0 ~ 8.0, mixes after adding solid activator, obtains initial mixing solution; (2) in initial mixing solution, add the phosphatide mixing of variable concentrations, coordinate calcium chloride solution to carry out Quality Control mensuration on Blood coagulation instrument, measurement result is exactly final proportioning closest to the ratio of reagents of the Quality Control scope intermediate value of this type Blood coagulation instrument; (3) according to final reagent proportioning, in initial mixing solution, add congealed fat, after stirring and evenly mixing, add polyglycol and stabilizing agent, after mixing, be the phospholipid reagent in APTT kit; (4) weigh lime chloride dissolution of solid in one-level water, mix the lime chloride reagent be in APTT kit; (5) APTT kit is obtained after phospholipid reagent and lime chloride reagent being packed respectively.
Beneficial effect of the present invention: a kind of APTT kit and preparation method thereof is provided, this APTT kit eliminates the step such as packing and freeze-drying in process for preparation, difference between the bottle avoiding the reagent caused because of freeze-drying and redissolution process, thus cause the larger difference between measurement result.Reagent of the present invention is without the need to freeze-drying, and get final product the stability of guarantee reagent, after ensure that uncork, the optimum validity of reagent continues at least one moon, avoid the larger difference between experimental result, save the consumption of reagent, uncork and use simultaneously, operated more fast and simple.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
APTT kit of the present invention, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different containers;
Phospholipid reagent is made up of solid activator, phosphatide and buffer system, the content of solid activator is 0.01 ~ 2g/L, the content of phosphatide is 2.5 ~ 8wt%, the pH of buffer system is 6.0 ~ 8.0, and in buffer system, each component concentration is: the polyglycol of the buffering agent of 0.1 ~ 1M, the stabilizing agent of 0.01 ~ 0.2wt%, 0.1 ~ 1.0wt%, all the other are water; Described buffering agent is selected from: Tris-Hcl, Hepes, PBS, MPOS; Described stabilizing agent is selected from: Sodium azide, Kathon, Proclin, glycocoll, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid;
The working concentration of lime chloride reagent is 15 ~ 40mM.
Prepare the method for above-mentioned APTT kit, comprise following step
(1) prepare buffer system, pH is adjusted to 6.0 ~ 8.0, mixes after adding solid activator, obtains initial mixing solution; (2) in initial mixing solution, add the phosphatide mixing of variable concentrations, coordinate calcium chloride solution to carry out Quality Control mensuration on Blood coagulation instrument, measurement result is exactly final proportioning closest to the ratio of reagents of the Quality Control scope intermediate value of this type Blood coagulation instrument; (3) according to final reagent proportioning, in initial mixing solution, add congealed fat, after stirring and evenly mixing, add polyglycol and stabilizing agent, after mixing, be the phospholipid reagent in APTT kit; (4) weigh lime chloride dissolution of solid in one-level water, mix the lime chloride reagent be in APTT kit; (5) APTT kit is obtained after phospholipid reagent and lime chloride reagent being packed respectively.
Embodiment 1
APTT kit, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels; Phospholipid reagent is made up of ellagic acid, phosphatide and buffer system, the content of ellagic acid is 2g/L, the content of phosphatide is 2.5wt%, and in buffer system, each component concentration is: the polyglycol of Kathon, 1.0wt% of Tris-Hcl, 0.01wt% of 1M, all the other are water, and the pH of buffer system is 6.0; The working concentration of lime chloride reagent is 15mM.When using kit of the present invention, the volume ratio of test plasma and phospholipid reagent consumption is 1:0.85 ~ 1.1.
Prepare the method for above-mentioned APTT kit, comprise following step
(1) prepare buffer system, pH is adjusted to 6.0 ~ 8.0, mixes after adding solid activator, obtains initial mixing solution; (2) in initial mixing solution, add the phosphatide mixing of variable concentrations, coordinate calcium chloride solution to carry out Quality Control mensuration on Blood coagulation instrument, the ratio of reagents of the measurement result Quality Control scope intermediate value of this type Blood coagulation instrument is exactly final proportioning; (3) according to final reagent proportioning, in initial mixing solution, add congealed fat, after stirring and evenly mixing, add polyglycol and stabilizing agent, after mixing, be the phospholipid reagent in APTT kit; (4) weigh lime chloride dissolution of solid in one-level water, mix the lime chloride reagent be in APTT kit; (5) APTT kit is obtained after phospholipid reagent and lime chloride reagent being packed respectively.
The kit that application the present embodiment obtains measures the prothrombin time of test plasma by semi-automatic coagulo meter, assay method is: first balance phospholipid reagent to room temperature, the bath of lime chloride reagent temperature is to 37 DEG C, again test plasma and phospholipid reagent are mixed according to the ratio that volume ratio is 1:0.85 ~ 1.1, be placed in 37 DEG C of temperature bath 3min, then add lime chloride reagent, make its final concentration be 15 ~ 40mM, mix timing at once, record prothrombin time.Do ten parallel laboratory tests, record data, analyze the repeatability of the present embodiment reagent, and in the present embodiment, reagent repeatability is as table 1.
Embodiment 2
APTT kit, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels; Described phospholipid reagent is made up of bentonite, rabbit cephalin and buffer system, the content of bentonite is 0.05g/L, the content of rabbit cephalin is 7wt%, and in buffer system, the content of each component is: the Sodium azide of MPOS, 0.1wt% of 0.4M, the polyglycol of 0.8wt%, all the other are water; The pH of buffer system is 7.8, and the working concentration of lime chloride reagent is 20mM.
APTT kit preparation method is with embodiment 1.
The kit that application the present embodiment obtains measures the prothrombin time of test plasma on semi-automatic coagulo meter, assay method, with embodiment 1, does ten parallel laboratory tests, record data, analyze the repeatability of the present embodiment reagent, in the present embodiment, reagent repeatability is as table 1.
Embodiment 3
APTT kit, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels; Phospholipid reagent is made up of solid activator, rabbit cephalin and buffer system, the content of solid activator is 1.0g/L, the content of rabbit cephalin is 5wt%, and in buffer system, the content of each component is: the stabilizing agent of Hepes, 0.05wt% of 0.75M, the polyglycol of 0.3wt%, all the other are water; The pH of buffer system is 6.0, and the working concentration of lime chloride reagent is 35mM; Wherein, solid activator is ellagic acid, porcelain earth and aluminium-magnesium silicate quality are the potpourri of 1:0.5:2, and stabilizing agent is the potpourri of Kathon, glycocoll, sero-abluminous 1:0.86:1.1.
APTT kit preparation method is with embodiment 1.
The kit that application the present embodiment obtains measures the prothrombin time of test plasma on semi-automatic coagulo meter, assay method, with embodiment 1, does ten parallel laboratory tests, record data, analyze the repeatability of the present embodiment reagent, in the present embodiment, reagent repeatability is as table 1.
Embodiment 4
APTT kit, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels; Phospholipid reagent is made up of zeyssatite, rabbit cephalin and buffer system, diatomaceous content is 1.5g/L, the content of rabbit cephalin is 3wt%, and in buffer system, the content of each component is: the polyglycol of Proclin, 0.9wt% of PBS, 0.03wt% of 0.25M, all the other are water; The pH of buffer system is 7.0, and the working concentration of lime chloride reagent is 18mM.
APTT kit preparation method is with embodiment 1.
The kit that application the present embodiment obtains measures the prothrombin time of test plasma on semi-automatic coagulo meter, assay method, with embodiment 1, does ten parallel laboratory tests, record data, analyze the repeatability of the present embodiment reagent, in the present embodiment, reagent repeatability is as table 1.
Embodiment 5
APTT kit, comprises phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels; Phospholipid reagent is made up of aluminium-magnesium silicate, rabbit cephalin and buffer system, the content of aluminium-magnesium silicate is 1.5g/L, the content of rabbit cephalin is 8wt%, and in buffer system, the content of each component is: the carbolic acid of Tris-Hcl, 0.2wt% of 0.1M, the polyglycol of 0.1wt%, all the other are water; The pH of buffer system is 8.0, and the working concentration of lime chloride reagent is 40mM.
APTT kit preparation method is with embodiment 1.
The kit that application the present embodiment obtains measures the prothrombin time of test plasma on semi-automatic coagulo meter, assay method, with embodiment 1, does ten parallel laboratory tests, record data, analyze the repeatability of the present embodiment reagent, in the present embodiment, reagent repeatability is as table 1.
Experimental example
Experimental example 1
The kit that Application Example 1 ~ 5 obtains measures the prothrombin time of test plasma by semi-automatic coagulo meter, assay method is: first balance phospholipid reagent to room temperature, the bath of lime chloride reagent temperature is to 37 DEG C, again test plasma and phospholipid reagent are mixed according to the ratio that volume ratio is 1:1, be placed in 37 DEG C of temperature bath 3min, then add lime chloride reagent, make its final concentration be 25mM, mix timing at once, record prothrombin time.The kit of each embodiment does ten parallel laboratory tests, record data.Measure same plasma sample with commercial reagent, record prothrombin time, reagent of the present invention and commercial reagent repeatability comparative result are as table 1.
Table 1: reagent of the present invention compares with commercial reagent repeatability
Table 1 shows, reagent of the present invention is compared with commercial reagent, and the coefficient of variation (CV) value is less, reproducible
Experimental example 2
The relatively stability of reagent of the present invention and commercial reagent, by reagent of the present invention and commercial reagent uncork simultaneously, commercial reagent is use after freeze-dried powder redissolves, reagent of the present invention directly uses, identical Blood coagulation instrument does Quality Control test, record the Quality Control result with the change on date reagent of the present invention and commercial reagent after uncork, the two stability compares as table 2.
Table 2: reagent of the present invention compares 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, the prothrombin time value of reagent of the present invention had no significant change in 40 days, and the prothrombin time value of commercial reagent just there occurs change from the 7th day, and along with the increase of number of days, its prothrombin time value, in obviously becoming large trend, illustrates that the stability of reagent of the present invention is better.
Claims (10)
1. an APTT kit, is characterized in that: comprise phospholipid reagent and lime chloride reagent, and two kinds of reagent are stored in different vessels;
Described phospholipid reagent is mixed by solid activator, phosphatide and buffer system; The content of described solid activator is 0.01 ~ 2g/L, and described solid activator to be selected from ellagic acid, bentonite, porcelain earth, zeyssatite, aluminium-magnesium silicate one or more potpourri; The content of described phosphatide is 2.5 ~ 8wt%; The pH of described buffer system is 6.0 ~ 8.0, and in described buffer system, each component concentration is: the polyglycol of the buffering agent of 0.1 ~ 1M, the stabilizing agent of 0.01 ~ 0.2wt%, 0.1 ~ 1.0wt%, all the other are water; Described buffering agent is selected from: Tris-Hcl, Hepes, PBS, MPOS; Described stabilizing agent is selected from: Sodium azide, Kathon, Proclin, glycocoll, bovine serum albumin(BSA), thimerosal, mercuric nitrate benzene, chloroform, carbolic acid;
The working concentration of described lime chloride reagent is 15 ~ 40mM.
2. APTT kit according to claim 1, is characterized in that: the content of described solid activator is 0.05 ~ 1g/L.
3. APTT kit according to claim 1, is characterized in that: described solid activator is ellagic acid, porcelain earth and aluminium-magnesium silicate quality are the potpourri of 1:0.5:2.
4. APTT kit according to claim 1, is characterized in that: described content of phospholipid is 5 ~ 7wt%.
5. the APTT kit according to claim 1 or 4, is characterized in that: described phosphatide is rabbit cephalin.
6. APTT kit according to claim 1, is characterized in that: the content of described buffering agent in buffer system is 0.4 ~ 0.75M.
7. APTT kit according to claim 1, is characterized in that: the content of described stabilizing agent in buffer system is 0.05 ~ 0.1wt%.
8. APTT kit according to claim 1, is characterized in that: the content of described polyglycol in buffer system is 0.3 ~ 0.8wt%.
9. APTT kit according to claim 1, is characterized in that: the working concentration of described lime chloride reagent is 20 ~ 35mM.
10. prepare the method for APTT kit described in claim 1, it is characterized in that: comprise the steps: (1) preparation buffer system, pH is adjusted to 6.0 ~ 8.0, mixes after adding solid activator, obtains initial mixing solution; (2) in initial mixing solution, add the phosphatide mixing of variable concentrations, coordinate calcium chloride solution to carry out Quality Control mensuration on Blood coagulation instrument, measurement result is exactly final proportioning closest to the ratio of reagents of the Quality Control scope intermediate value of this type Blood coagulation instrument; (3) according to final reagent proportioning, in initial mixing solution, add congealed fat, after stirring and evenly mixing, add polyglycol and stabilizing agent, after mixing, be the phospholipid reagent in APTT kit; (4) weigh lime chloride dissolution of solid in one-level water, mix the lime chloride reagent be in APTT kit; (5) APTT kit is obtained after phospholipid reagent and lime chloride reagent being packed respectively.
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| CN106290923A (en) * | 2016-08-01 | 2017-01-04 | 重庆鼎润医疗器械有限责任公司 | A kind of activation coagulation assay reagent and application thereof |
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| CN107748267A (en) * | 2017-10-13 | 2018-03-02 | 山东艾科达生物科技有限公司 | One kind measure activated partial thromboplastin time（APTT）Kit |
| 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 |
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| 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 |
| CN110412001A (en) * | 2019-08-01 | 2019-11-05 | 武汉塞力斯生物技术有限公司 | A kind of kit of dynamic monitoring fibrin ferment generative capacity |
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| CN106290923A (en) * | 2016-08-01 | 2017-01-04 | 重庆鼎润医疗器械有限责任公司 | A kind of activation coagulation assay reagent and application thereof |
| 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 |
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