CN103197083A - D-dimer quality control product and preparation method thereof - Google Patents
D-dimer quality control product and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of quality control of clinical blood coagulation detection projects, and in particular relates to a method for preparing a D-dimer quality control product. The method comprises the following steps: carrying out recalcification on bovine plasma and solidifying; adding an enzyme capable of degrading fibrous protein, thereby dissolving a fibrous protein clot; stopping the reaction by using a protease inhibitor; diluting a generated D-dimer mother liquor by using a freeze-dried protecting solution; and freeze-drying, thereby obtaining the D-dimer quality control product. The prepared D-dimer quality control product is very sensitive to the change of a detection reagent and good in stability, thereby satisfying the quality control requirement of clinical D-dimer detection.
Description
Technical field
The present invention relates to clinical detection of blood coagulation project quality control field, particularly a kind of method that adopts the ox blood slurry to prepare D-dimer quality-control product.
Background technology
Fibrinogen is the highest albumen of content in the mammalian plasma, has high homology between different mammals, and the fibrin coagula forms after fibrinogen is activated in animal body, blood clotting.In blood clotting, activated fibrinolytic system, it is the most important blood anticoagulant of human body system: when fibrin condenses, the plasminogen activator plasminogen activation is converted into fibrinolysin, the fibrinolysis process begins, fibrinolysin fibrin degradation coagula forms various solvable fragments, form fibrin degradation product (FDP) (FDP), FDP by: X, Y, D-dimer, intermediate segment E fragment etc. are formed.
The D-dimer is the minimal segment in the fibrin degradation product (FDP), it also is the specificity catabolite of crosslinked fibrin, it is the molecular marker of reflection blood coagulation and fibrinolytic activation, the D-dimer content is very little among the human normal plasma, but can cause all in the numerous disease that forms with thrombus and microthrombus that D-is dimeric to be increased, so it is one of important sensitive indicator of hypercoagulative state and secondary hyperfibrinolysis in the antimer at present, is applicable to the monitoring thrombosis too, fibrinolytic enhancing and thromboembolism treatment.
Along with D-dimer Interventions Requested constantly are extended to every field in medical diagnosis on disease and the treatment, use supporting D-dimer quality-control product that the D-dimer is checked and carry out indoor quality control and also seem particularly important.Quality-control product is external imported product in the D-dimer measuring cell of present domestic application, and is expensive.Also have bibliographical information to detect back residue blood plasma with experiment and prepare D-dimer quality-control product, but the raw material sources that this method is used are human plasma, are difficult for obtaining, and are not suitable for commercialization production.
Summary of the invention
In view of this, the invention provides a kind of ox blood slurry that adopts and prepare the method for D-dimer quality-control product and the D-dimer quality-control product of acquisition.The D-dimer quality-control product of the inventive method preparation, good to detecting reagent variation susceptibility, and have stability preferably, satisfy clinical D-dimer inspection quality control requirement.
In order to realize the foregoing invention purpose, the invention provides following technical scheme:
The invention provides a kind of method of the D-of preparation dimer quality-control product; getting the ox blood slurry mixes with ionic calcium soln; 37 ℃ of water-baths make blood clotting; the enzyme that adds biodegradable fiber albumen again, 37 ℃ of water-baths made the fibrin clot dissolving in 4～8 hours, used the inhibitor cessation reaction; obtain D-dimer mother liquor; use frozen-dried protective liquid dilution mother liquor, freeze drying obtains D-dimer quality-control product.
In preparation method provided by the invention, the preparation method of ox blood slurry be the blood of gathering with bovine jugular vein, with blood by with anti-coagulants be 9:1 adding anti-coagulants with the volume ratio, centrifugal 30 minutes, relative centrifugal force(RCF) is 2000～2500g, and careful separation is drawn upper plasma, namely; Anti-coagulants is 2～5% trisodium citrates.
In preparation method provided by the invention, ionic calcium soln is 0.01～0.05mol/L calcium chloride solution, and addition is 30%～70% of ox blood volume of slurry.
In preparation method provided by the invention, the enzyme of biodegradable fiber albumen is any one in streptokinase, the fibrinolysin.
As preferably, the enzyme of biodegradable fiber albumen is fibrinolysin, adds that the final concentration of fibrinolysin is 100～300U/mL after the reaction system.
As preferably, inhibitor is Aprotinin, adds that the final concentration of Aprotinin is 0.1～1.0 μ g/mL after the reaction system.
In preparation method provided by the invention; frozen-dried protective liquid comprises 3-Pehanorm-2-hydroxy-propanesulfonic acid (TAPSO) 1～3%(w/v), sodium chloride 1～5%(w/v), Sodium azide 0.5～3g/L, sweet mellow wine 5～20g/L, bovine serum albumin(BSA) 10～30g/L, Aprotinin 10000～100000KIU/L.
The D-dimer quality-control product that the present invention also provides method for preparing to obtain.
The present invention takes above technical scheme, compared with prior art, has the following advantages:
1, the present invention selects for use the ox blood slurry to be feedstock production D-dimer quality-control product, and cost of material is low, the source is sufficient, be easy to acquisition, and preparation technology of the present invention is simple, and the production cost is low.
2, the quality-control product of the inventive method preparation change sensitivity to detecting reagent, and dried frozen aquatic products redissolution rear stability is better, alternative imported product is used for the quality control that the D-dimer detects, and the holding time prolongs, and saves and uses reagent, reduce and detect cost, bring convenience to work.
Description of drawings
Fig. 1 shows D-dimer quality-control product and the commercially available D-dimer uncork Detection of Stability result of the embodiment of the invention 1 preparation; Wherein, curve 1 shows the quality-control product that the embodiment of the invention 1 prepares; Curve 2 shows control group Siemens quality-control product.
Embodiment
The invention discloses a kind of ox blood slurry that adopts and prepare the method for D-dimer quality-control product and the D-dimer quality-control product of acquisition, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
Employing ox blood provided by the invention slurry prepares in the D-dimer quality-control product of the method for D-dimer quality-control product and acquisition raw materials used and reagent and all can be buied by market.
Below in conjunction with embodiment, further set forth the present invention:
The preparation of embodiment 1D-dimer quality-control product
1, ox blood slurry: the blood that will gather from bovine jugular vein, add 3.8% trisodium citrate anti-coagulants solution, the volume ratio of blood and anti-coagulants is 9:1.To add the centrifugal blood 30 minutes of anti-coagulants, relative centrifugal force(RCF) is 2000g, and careful separation is drawn upper plasma.
2, freeze drying protectant: TAPSO1.3%, sodium chloride 2%, Sodium azide 1.0g/L, sweet mellow wine 10g/L, bovine serum albumin(BSA) 20g/L, Aprotinin 80000KIU/L.
3, D-dimer quality-control product preparation:
(1) ox blood slurry is divided install in the glass test tube, every pipe adds the calcium chloride solution of the 0.025mol/L of plasma volume 40%, and 37 ℃ of water-baths 15 minutes are until the clotting of plasma.
(2) adding fibrinolysin solution in the blood plasma, to make its final concentration be 240U/mL, was positioned over 37 ℃ of water baths 6 hours, stirred once with glass bar every one hour therebetween, all dissolves until fibrin clot.
(3) adding Aprotinin solution, to make its final concentration be 0.48 μ g/mL, and cessation reaction produces D-dimer mother liquor.
(4) mother liquor is diluted with the 1:100 ratio with frozen-dried protective liquid, press the packing of 0.5ml/ bottle, freeze drying obtains D-dimer quality-control product, measures with the latex immunoturbidimetry, and definite value is 32.1 μ g/mL.
Embodiment 2D-dimer quality-control product to the reagent changing sensitivity relatively
1, the D-dimer quality-control product of the D-dimer quality-control product of preparation among the embodiment 1 and commercially available German Siemens company is accurately redissolved reconstruction.
2, the D-dimer detection kit of the German Siemens of use company is carried out the detection of D-dimer, and detection method is the latex immunoturbidimetry.With R in the kit
2Reagent (immune emulsion reagent) carries out serial dilution with distilled water, obtains R
2Concentration is: 80%, 60%, 40%, 20%, 10% reagent 100%.
3, on the full-automatic coagulo meter of Japanese East Asia CA1500, use the R of serial dilution respectively
2Other reagent in reagent and the kit detects the D-dimer quality-control product and the commercially available German Siemens D-of the company dimer quality-control product that prepare among the embodiment 1, and testing result is as shown in table 1.The result shows: detect reagent R
2Concentration from 100% to 10%, D-dimer quality-control product testing result variation range of the present invention is bigger, and is higher to the reagent changing sensitivity, shows that quality-control product of the present invention has higher susceptibility in the monitoring to defectiveness reagent.
Table 1 reagent variation sensitivity detects
Embodiment 3D-dimer quality-control product Detection of Stability.
1, uncork Detection of Stability
D-dimer quality-control product and the German Siemens company quality-control product of preparation among the embodiment 1 are accurately redissolved respectively, under room temperature (15～25 ℃) condition, preserve, every day timing sampling one-time detection D-dimer quality-control product concentration.Adopt return law of the straight line to analyze the stability of Quality Control thing, the slope b and 0 of check linear equation has there was no significant difference, as P＜0.05, illustrates that analyte concentration changes in time, as P＞0.05, illustrates that analyte concentration is basicly stable in minute.Testing result such as table 2 and shown in Figure 1.
Table 2 uncork Detection of Stability result
Quality-control product regression equation of the present invention is: y=-0.0182x+31.882, carry out the sided t check to the significant difference of slope b and 0: α=0.05 as can be known of tabling look-up, ν=9, t
α (ν)=2.262; According to statistical calculations, t
b=-0.124, P＞0.05 shows that storing 10 days at ambient temperature after D-dimer quality-control product that the present invention prepares redissolves is stable substantially.Siemens quality-control product regression equation is: y=-0.5155x+32.586, t
b=-2.847, P＜0.05, show commercially available quality-control product at ambient temperature uncork store 10 days, obvious variation appears in D-dimer concentration, and as seen from Figure 1, obviously decline occurs since the 8th day D-dimer concentration.
This shows, the D-dimer of the present invention's preparation is compared with the commercially available prod, has better uncork stability, for the less basic hospital of hospital clinical clinical laboratory, particularly portfolio, in the Quality Control work that the D-dimer detects, preserve the longer time after the quality-control product uncork, can reduce reagent waste, reduce and detect cost, bring convenience to work.
2, long-time stability detect
The D-dimer quality-control product dried frozen aquatic products of preparation among the embodiment 1 is preserved under+2～+ 8 ℃ of conditions, taken out one bottle of accurately redissolution in per 3 months, detect D-dimer quality-control product concentration, testing result is as shown in table 3.
Table 3 long-time stability testing result
By the return law of the straight line analysis, quality-control product regression equation of the present invention is: y=-0.0288x+31.259, carry out the sided t check to the significant difference of slope b and 0: α=0.05 as can be known of tabling look-up, ν=10, t
α (ν)=2.228; According to statistical calculations, t
b=-1.570, P＞0.05, show D-dimer quality-control product that the present invention prepares+2～+ 8 ℃ of conditions preserve 33 months basicly stable, long-time stability are better, are conducive to commercialization production.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. method for preparing D-dimer quality-control product; it is characterized in that; get the ox blood slurry and mix with ionic calcium soln, 37 ℃ of water-baths make blood clotting, add the enzyme of biodegradable fiber albumen again; 37 ℃ of water-baths made the fibrin clot dissolving in 4～8 hours; use the inhibitor cessation reaction, obtain D-dimer mother liquor, use frozen-dried protective liquid dilution mother liquor; freeze drying obtains D-dimer quality-control product.
2. method according to claim 1, it is characterized in that, the preparation method of described ox blood slurry is the blood of gathering with bovine jugular vein, with blood by with anti-coagulants be that 9:1 adds anti-coagulants with the volume ratio, centrifugal 30 minutes, relative centrifugal force(RCF) is 2000～2500g, and careful separation is drawn upper plasma, namely; Described anti-coagulants is 2～5% trisodium citrates.
3. method according to claim 1 is characterized in that, described ionic calcium soln is 0.01～0.05mol/L calcium chloride solution, and addition is 30%～70% of described ox blood volume of slurry.
4. method according to claim 1 is characterized in that, the enzyme of described biodegradable fiber albumen is any one in streptokinase, the fibrinolysin.
5. method according to claim 4 is characterized in that, the enzyme of described biodegradable fiber albumen is fibrinolysin, and the final concentration of described fibrinolysin is 100～300U/mL after the adding reaction system.
6. method according to claim 1 is characterized in that, described inhibitor is Aprotinin, and the final concentration of described Aprotinin is 0.1～1.0 μ g/mL after the adding reaction system.
7. method according to claim 1; it is characterized in that; described frozen-dried protective liquid comprises 3-Pehanorm-2-hydroxy-propanesulfonic acid 1～3%(w/v), sodium chloride 1～5%(w/v), Sodium azide 0.5～3g/L, sweet mellow wine 5～20g/L, bovine serum albumin(BSA) 10～30g/L, Aprotinin 10000～100000KIU/L.
8. the D-dimer quality-control product for preparing according to each described method of claim 1 to 7.
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Cited By (10)
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| CN104459103A (en) * | 2014-12-15 | 2015-03-25 | 中国医学科学院输血研究所 | Preparation method of D-dimer quality controls |
| CN104458367A (en) * | 2014-11-06 | 2015-03-25 | 上海长岛生物技术有限公司 | D-dimer and FDP (Fibrin/Fibringen Degradation Product) composite quality control material and preparation method thereof |
| CN104569410A (en) * | 2015-01-04 | 2015-04-29 | 深圳市艾瑞生物科技有限公司 | Homogeneous fluorescence immunoassay reagent group for rapidly and quantitatively detecting D-dimer and preparation method of homogeneous fluorescence immunoassay reagent group |
| CN104880350A (en) * | 2015-05-22 | 2015-09-02 | 宁波美康生物科技股份有限公司 | Preparation method for leucine aminopeptidase quality control product |
| CN107677839A (en) * | 2017-09-20 | 2018-02-09 | 北京众驰伟业科技发展有限公司 | A kind of preparation method of the compound quality-control product of D dimers and FDP |
| CN107677528A (en) * | 2017-09-20 | 2018-02-09 | 上海贞元诊断用品科技有限公司 | Calibration object and quality-control product of a kind of anticoagulant heparin detection reagent and preparation method thereof |
| CN109298176A (en) * | 2018-10-29 | 2019-02-01 | 深圳天深医疗器械有限公司 | Myocarditis quality-control product and preparation method thereof, myocarditis detection kit and myocarditis detection device |
| CN110684817A (en) * | 2019-10-23 | 2020-01-14 | 北京赛科希德科技股份有限公司 | Human fibrin in vitro degradation method |
| CN111337694A (en) * | 2020-03-07 | 2020-06-26 | 苏州德沃生物技术有限公司 | Quality control product of fibrinogen degradation product and preparation method thereof |
| CN114133445A (en) * | 2021-12-01 | 2022-03-04 | 北京赛科希德科技股份有限公司 | Preparation method of fibrinogen degradation fragment |
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| CN104458367A (en) * | 2014-11-06 | 2015-03-25 | 上海长岛生物技术有限公司 | D-dimer and FDP (Fibrin/Fibringen Degradation Product) composite quality control material and preparation method thereof |
| CN104458367B (en) * | 2014-11-06 | 2019-03-26 | 上海长岛生物技术有限公司 | A kind of d-dimer and the compound quality-control product of FDP and preparation method thereof |
| CN104459103A (en) * | 2014-12-15 | 2015-03-25 | 中国医学科学院输血研究所 | Preparation method of D-dimer quality controls |
| CN104569410A (en) * | 2015-01-04 | 2015-04-29 | 深圳市艾瑞生物科技有限公司 | Homogeneous fluorescence immunoassay reagent group for rapidly and quantitatively detecting D-dimer and preparation method of homogeneous fluorescence immunoassay reagent group |
| CN104880350A (en) * | 2015-05-22 | 2015-09-02 | 宁波美康生物科技股份有限公司 | Preparation method for leucine aminopeptidase quality control product |
| CN104880350B (en) * | 2015-05-22 | 2017-11-28 | 美康生物科技股份有限公司 | A kind of preparation method of leucine aminopeptidase quality-control product |
| CN107677528A (en) * | 2017-09-20 | 2018-02-09 | 上海贞元诊断用品科技有限公司 | Calibration object and quality-control product of a kind of anticoagulant heparin detection reagent and preparation method thereof |
| CN107677839A (en) * | 2017-09-20 | 2018-02-09 | 北京众驰伟业科技发展有限公司 | A kind of preparation method of the compound quality-control product of D dimers and FDP |
| CN107677839B (en) * | 2017-09-20 | 2020-11-17 | 北京众驰伟业科技发展有限公司 | Preparation method of D-dimer and FDP composite quality control product |
| CN109298176A (en) * | 2018-10-29 | 2019-02-01 | 深圳天深医疗器械有限公司 | Myocarditis quality-control product and preparation method thereof, myocarditis detection kit and myocarditis detection device |
| CN110684817A (en) * | 2019-10-23 | 2020-01-14 | 北京赛科希德科技股份有限公司 | Human fibrin in vitro degradation method |
| CN111337694A (en) * | 2020-03-07 | 2020-06-26 | 苏州德沃生物技术有限公司 | Quality control product of fibrinogen degradation product and preparation method thereof |
| CN114133445A (en) * | 2021-12-01 | 2022-03-04 | 北京赛科希德科技股份有限公司 | Preparation method of fibrinogen degradation fragment |
| CN114133445B (en) * | 2021-12-01 | 2023-11-17 | 北京赛科希德科技股份有限公司 | Preparation method of fibrinogen degradation fragment |
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