D-二聚体质控品制备方法学研究

Development of Stable Quality Control Plasma of D-Dimmer

目的研糊稳定的D-二聚体质控品，用于实验室内部D-二聚体检测的质量控制。方法①选择合适的原料制备D二聚体质控品。分别用肝素、枸橼酸钠和EDTA-K2抗凝的三种不同抗凝血为原料，都使用尿激酶为裂解酶，分别制备得到D-二聚体，比较其效价水平。②选择合适的纤维蛋白裂解酶来制备D-二聚体质控品。将EDTA-K2抗凝血作为原料，分别使用三种不同的纤维蛋白裂解酶-链激酶、尿激酶和纤维蛋白溶解酶，比较其裂解效果。③选择稳定的D-二聚体质控品介质组分。三种候选介质组分别在4℃和37℃保存条件下连续检测5天，以确定其稳定性。④对研制的D-二聚体质控品进行37℃，4℃和-35℃保存条件下的稳定性测试。⑤用研制的D-二聚体质控品对不同批号及不同检测系统D-二聚体试剂盒进行检测，以建立实验室内部D-二聚体检测的质量控制方法。结果①相同条件下，肝素、枸橼酸钠和EDTA—K2三种不同抗凝血刺备D-二聚体质控品的水平分别是24mg／L，75mg／L和100mg／L。因此选择效价最高的EDTA-K2抗凝血浆作为制备D-二聚体质控品的原料。②用链激酶、尿激酶和纤维蛋白溶解酶裂解纤维蛋白，分别得D-二聚体60mg／L，110mg／L和50mg／L。因此选择尿激酶作为制备D-二聚体质控品的裂解酶。③由25mmol／L pH7．4磷酸缓冲液（PBS），60g／L蔗糖，20g／L聚乙二醇2万衍生物，10g／L小牛血清清蛋白（BSA），0．5g／L叠氮钠（NaN3）组成的D-二聚体质控品的介质组分最稳定。④7．0mg／L浓度质控品，在37℃下，至少稳定5天，4℃可以保存4个月，-35℃冷冻保存到目前试验阶段已10个月。⑤用不同批号试剂盒检测系列浓度质控品，实际测定值与预期值的相关系数r≥0．95。不同检测系统对D-二聚体质控品的测定差异较大，因此，实验室内部研制的D-二聚体质控品不适用于室间质控。结论实验室内部的D-二聚体质控品可用健康人EDTA-K2抗凝血浆为原料，用尿激酶降解后，由25mmol／L pH7．4磷酸缓冲液（PBS），60g／L蔗糖，20g／L聚乙二醇2万衍生物，10g／L小牛血清清蛋白（BSA），0．5g／L叠氮钠（NaN3）作为介质组分，定标制备。该质控品具有良好的稳定性，可作为实验室内部D-二聚体检测的质量控制，以弥补因缺乏D-二聚体国际和国家标准品带来的测试结果缺少可靠性控制的问题。

Objective To develop stable quality control plasma of D-dimer,which is used in the quality control in the D-dimmer detection in the laboratory. Methods （1）To choose appropriate raw materials to produce quality control plasma of D-dimer and,by using urokinase as the fibrin repressible enzyme, used three different anticoagulation plasma of heparin, folic acid sodium,and EDTA-Kz anticoagulation respectively,which were disintegrated into dense dimmer to produce its quality control plasma and compared the differences among them, （2）To choose appropriate fibrin repressible enzyme to produce quality control plasma of D-dimer and use EDTA-K2 anticoagulation as the raw material. To analyze the different disintegrating effects on fibrin clots pyrolysis by three kinds of fibrin repressible enzyme-urokinase, streptokinase and fibrinolysis enzyme. （3）To choose stable medium composition of the quality control plasma of D-dimer and the three medium compositions to be chosen will be stored under the temperature of 4℃ and 37℃ for five days respectively to study its stability. （4）To check the stability of the quality control plasma of D-dimer produced by storing it under the temperature of 37℃,4℃ and -35℃ respectively. （5）To examine the D-dimer testing reagents of different batch numbers and testing systemswith the quality control plasma of D-dimer produced to establish the quality control method of testing the D-dimer in the laboratory. Results （1）in the same situation, the potency of quality control plasma of D-dimer made by three different anticoagulants of Heparin,folic acid sodium,and EDTA-K2 were 24 mg/L, 75 mg/L and 100 mg/L respectively. So,the anticoagulant of EDTA-K2 was chosen as the raw material in producing the quality control plasma of D-dimer. （2）The potency of quality control plasma of D-dimer made by disintegrating fibre protein with three different fibrin repressible enzymes of streptokinase,urokinase and fibrinolysis enzyme were 60 mg/L, 110 mg/L and 50 mg/L respectively. So, urokinase was chosen to produce the disintegrating enzyme of quality control plasma of D-dimer. （3）D-dimer medium composition ,which includes 25 mmol/L pH7. 4 PBS,60 g/L of sucrose,20 g/L of Carbowax,10 g/L of BSA dialysis content and 0. 5 mg/L of D-NaN3 ,was the most stable. （3）When its concentration was 7. 0 mg/L,it could remain stable for 5 to 7 days below 37 ℃. When the storage temperature was 4 ℃, it could be stored up to four months. The test of storing it under the temperature of -35℃ had been kept for 10 months by now. （5）To use testing reagents of different batch numbers to test the quality control plasma of different concentrations, the related coefficient r defining the actual testing statistics versus the anticipated statistics was equal or more than 0. 95. The differences among testing results on the quality control plasma by different testing systems were quite varied. Thus, the quality control plasma of D-dimer produced in the laboratory was not suitable for the different laboratory of the quality control. Conclusion After being decomposed by EDTA-K2 anticoagulation plasma and urokinase from the healthy body, the quality control plasma of D-dimer in the laboratory,whose medium composition includes 25 mmol/L pH7.4 PBS,60 g/L of sucrose, 20 g/L of Carbowax, 10 g/L of BSA dialysis content and 0. 5 mg/L of D-NaN3 ,could be made with set standard and remains stable. It could be used in quality control in detecting the D-dimer in the laboratory to make up for the unreliability of detection result due to lack of international-and national-standard D-dimer.