CN103197083B - 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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- CN103197083B CN103197083B CN201310090638.4A CN201310090638A CN103197083B CN 103197083 B CN103197083 B CN 103197083B CN 201310090638 A CN201310090638 A CN 201310090638A CN 103197083 B CN103197083 B CN 103197083B
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Abstract
The present invention relates to clinical detection of blood coagulation project integration field, specifically provide a kind of method preparing D-dimer quality control product.Method provided by the invention is that after being solidified by multiple for ox blood slurry calcium, the enzyme adding biodegradable fiber albumen makes fibrin clot dissolve, and uses protease inhibitors cessation reaction, and the DDi mother liquor frozen-dried protective liquid of generation dilutes, and freeze drying, to obtain final product.D-dimer quality control product prepared by the inventive method, good to detection reagent sensitive, and there is good stability, meet the quality control requirement of clinical DDi inspection.
Description
Technical field
The present invention relates to clinical detection of blood coagulation project integration field, particularly a kind of ox blood that adopts starches the method preparing D-dimer quality control product.
Background technology
Fibrinogen is the albumen that in mammalian plasma, content is the highest, between different mammals, have high homology, and after fibrinogen is activated, fibrin coagula is formed in animal body, blood clotting.Fibrinolytic system is have activated while blood clotting, it is the most important blood anticoagulant system of human body: when fibrin condensation, plasminogen activator plasminogen activation is converted into fibrinolysin, plasmin solution preocess starts, plasmin degradation fibrin coagula forms various solvable fragment, formed fibrin degradation product (FDP) (FDP), FDP by: X, Y, DDi, intermediate segment E fragment etc. form.
DDi is the minimal segment in fibrin degradation product (FDP), also be the selective degradation product of crosslinked fibrin, it is the molecular marker of reflection blood coagulation and fibrinolytic activation, in human normal plasma, DDi content is very micro-, but in the numerous disease with thrombus and microvascular corrosion cast, all can cause increasing of DDi, so it is one of important sensitive indicator of hypercoagulative state and secondary hyperfibrinolysis in antimer at present, be applicable to too monitor thrombosis, fibrinolytic enhancing and thromboembolism treatment.
Along with DDi Interventions Requested are constantly extended to the every field in medical diagnosis on disease and treatment, use supporting D-dimer quality control product to carry out indoor quality control to DDi inspection and also seem particularly important.The DDi of current domestic application measures Internal Quality Control product and is external imported product, expensive.Also have bibliographical information to test and detect rear residue blood plasma to prepare D-dimer quality control product, but the raw material sources that the method uses are human plasma, not easily obtain, are not suitable for merchandized handling.
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.D-dimer quality control product prepared by the inventive method, good to detection reagent sensitive, and there is good stability, meet the quality control requirement of clinical DDi inspection.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of method preparing D-dimer quality control product; get ox blood slurry to mix with ionic calcium soln; 37 DEG C of water-baths make blood clotting; add the enzyme of biodegradable fiber albumen again, 37 DEG C of water-baths make fibrin clot dissolve in 4 ~ 8 hours, use inhibitor cessation reaction; obtain DDi 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 is the blood gathered with bovine jugular vein, by blood by being that 9:1 adds anti-coagulants with anti-coagulants with volume ratio, centrifugal 30 minutes, relative centrifugal force(RCF) is 2000 ~ 2500g, and careful separation draws upper plasma, to obtain final product; 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, fibrinolysin.
As preferably, the enzyme of biodegradable fiber albumen is fibrinolysin, and after adding reaction system, the final concentration of fibrinolysin is 100 ~ 300U/mL.
As preferably, inhibitor is Aprotinin, and after adding reaction system, the final concentration of Aprotinin is 0.1 ~ 1.0 μ g/mL.
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.
Present invention also offers the D-dimer quality control product that said method prepares.
The present invention takes above technical scheme, compared with prior art, has the following advantages:
1, the present invention selects ox blood to starch as D-dimer quality control product prepared by raw material, and cost of material is low, source is sufficient, be easy to acquisition, and preparation technology of the present invention is simple, and production cost is low.
2, the quality-control product prepared of the inventive method, to detection reagent sensitive, and dried frozen aquatic products to redissolve rear stability better, alternative imported product is used for the quality control that DDi detects, and the holding time extends, and saves and uses reagent, reduce testing cost, bring convenience to work.
Accompanying drawing explanation
Fig. 1 shows D-dimer quality control product prepared by the embodiment of the present invention 1 and commercially available DDi uncork Detection of Stability result; Wherein, curve 1 shows the quality-control product that the embodiment of the present 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 present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Employing ox blood provided by the invention slurry is prepared raw materials used in the method for D-dimer quality control product and the D-dimer quality control product of acquisition and reagent and all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1D-dimer quality-control product
1, ox blood slurry: by the blood gathered 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 draws 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) be dispensed in glass test tube by ox blood slurry, often pipe adds the calcium chloride solution of the 0.025mol/L of plasma volume 40%, and 37 DEG C of water-baths 15 minutes are until the clotting of plasma.
(2) in blood plasma, adding fibrinolysin solution makes its final concentration be 240U/mL, is positioned over 37 DEG C of water baths 6 hours, stirs once, until fibrin clot all dissolves therebetween every one hour with glass bar.
(3) adding Aprotinin solution makes its final concentration be 0.48 μ g/mL, cessation reaction, produces DDi mother liquor.
(4) diluted with 1:100 ratio by mother liquor frozen-dried protective liquid, by the packing of 0.5ml/ bottle, freeze drying, obtain D-dimer quality control product, measure by latex immunoturbidimetry, definite value is 32.1 μ g/mL.
Embodiment 2D-dimer quality-control product compares reagent changing sensitivity
1, the D-dimer quality control product of preparation in embodiment 1 and the D-dimer quality control product of commercially available German Siemens company are accurately redissolved reconstruction.
2, use the DDi detection kit of German Siemens company to carry out DDi detection, detection method is latex immunoturbidimetry.By R in kit
2reagent (immune emulsion reagent) carries out serial dilution with distilled water, obtains R
2concentration is: 100%, 80%, 60%, 40%, 20%, 10% reagent.
3, on the CA1500 Automatic coagulometer of Japanese East Asia, use the R of serial dilution respectively
2other reagent in reagent and kit, detect D-dimer quality control product and the commercially available German Siemens company D-dimer quality control product of preparation in embodiment 1, testing result is as shown in table 1.Result shows: detect reagent R
2concentration is from 100% to 10%, and D-dimer quality control product testing result variation range of the present invention is larger, higher to reagent changing sensitivity, shows that quality-control product of the present invention has higher susceptibility to the monitoring of defectiveness reagent.
Table 1 reagent variation sensitivity technique
Embodiment 3D-dimer quality-control product Detection of Stability.
1, uncork Detection of Stability
The D-dimer quality control product of preparation in embodiment 1 and German Siemens company quality-control product are accurately redissolved respectively, preserve under room temperature (15 ~ 25 DEG C) condition, every day timing sampling one-time detection D-dimer quality control product concentration.Adopt the stability of return law of the straight line Analysis quality control thing, the slope b and 0 of inspection linear equation has there was no significant difference, as P < 0.05, illustrate that analyte concentration changes in time, as P > 0.05, illustrate that analyte concentration is basicly stable in minute.Testing result as shown in Table 2 and Figure 1.
Table 2 uncork Detection of Stability result
Quality-control product regression equation of the present invention is: y=-0.0182x+31.882, carries out sided t inspection to the significant difference of slope b and 0: known α=0.05 of tabling look-up, ν=9, t
α (ν)=2.262; According to statistical calculations, t
b=-0.124, P > 0.05, storing 10 days at ambient temperature after showing D-dimer quality control product redissolution prepared by the present invention 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, there is significantly change in DDi concentration, and as seen from Figure 1, from the 8th day s, obviously decline appears in DDi concentration.
As can be seen here, DDi prepared by the present invention is compared with commercially available prod, there is better uncork stability, for the basic hospital that hospital clinical clinical laboratory, particularly portfolio are less, in the Quality Control work that DDi detects, the longer time is preserved after quality-control product uncork, can reagent waste be reduced, reduce testing cost, bring convenience to work.
2, long-time stability detect
Preserved under+2 ~+8 DEG C of conditions by the D-dimer quality control product dried frozen aquatic products of preparation in embodiment 1, within every 3 months, take out one bottle and accurately redissolve, detect D-dimer quality control product concentration, testing result is as shown in table 3.
Table 3 long-time stability testing result
Analyzed by return law of the straight line, quality-control product regression equation of the present invention is: y=-0.0288x+31.259, carries out sided t inspection to the significant difference of slope b and 0: known α=0.05 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 prepared by the present invention+2 ~+8 DEG C of conditions preserve 33 months basicly stable, long-time stability are better, are conducive to merchandized handling.
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 premise without departing from the principles 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 (7)
1. prepare the method for D-dimer quality control product for one kind, it is characterized in that, get ox blood slurry to mix with ionic calcium soln, 37 DEG C of water-baths make blood clotting, then add the enzyme of biodegradable fiber albumen, 37 DEG C of water-baths make fibrin clot dissolve in 4 ~ 8 hours, use inhibitor cessation reaction, obtain DDi mother liquor, use frozen-dried protective liquid dilution mother liquor, freeze drying, obtains D-dimer quality control product; Wherein, 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.
2. method according to claim 1, it is characterized in that, the preparation method of described ox blood slurry is the blood gathered with bovine jugular vein, by blood by being that 9:1 adds anti-coagulants with anti-coagulants with volume ratio, centrifugal 30 minutes, relative centrifugal force(RCF) is 2000 ~ 2500g, and careful separation draws upper plasma, to obtain final product; 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, fibrinolysin.
5. method according to claim 4, is characterized in that, the enzyme of described biodegradable fiber albumen is fibrinolysin, and after adding reaction system, the final concentration of described fibrinolysin is 100 ~ 300U/mL.
6. method according to claim 1, is characterized in that, described inhibitor is Aprotinin, and after adding reaction system, the final concentration of described Aprotinin is 0.1 ~ 1.0 μ g/mL.
7. the D-dimer quality control product that the method according to any one of claim 1 to 6 prepares.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN104459103B (en) * | 2014-12-15 | 2016-04-27 | 中国医学科学院输血研究所 | The preparation method of D-dimer quality control product |
| CN104569410B (en) * | 2015-01-04 | 2017-01-11 | 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 |
| 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 |
| 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 |
| 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 |
| CN110684817B (en) * | 2019-10-23 | 2021-07-20 | 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 |
| 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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