抗凝剂对定量检测乙型肝炎病毒DNA的影响

Effect of anticoagulants on quantitative detection of HBV DNA

目的探讨用实时荧光定量PCR技术检测乙型肝炎病毒DNA拷贝数时,不同抗凝剂对乙型肝炎病毒DNA定量检测的影响。方法采集40例乙型肝炎患者血液,分别置于不含抗凝剂及含肝素钠、枸缘酸钠、EDTA-K2抗凝剂的无菌试管中,分离血清和血浆后于-20℃冰箱保存备用。采用实时荧光定量PCR方法检测血清和血浆中乙型肝炎病毒DNA的拷贝数,用配对t检验对检测结果进行统计处理。结果40例乙型肝炎患者不含抗凝剂组的乙型肝炎病毒DNA拷贝数的对数均数为5.66±2.10,含肝素钠组的对数均数为5.42±2.26,含枸缘酸钠组的对数均数为5.36±2.11,含EDTA-K2组的对数均数为5.58±2.16。三种含抗凝剂的标本分别与不含抗凝剂的血清组标本中的乙型肝炎病毒DNA拷贝数转换为对数经配对t检验统计处理,含肝素钠组和枸缘酸钠组的DNA拷贝数与不含抗凝剂组DNA拷贝数差异有统计学意义(P<0.05),而含EDTA-K2组的DNA拷贝数与不含抗凝剂组的的DNA拷贝数差异无统计学意义(P>0.05)。结论实时荧光定量PCR技术检测乙型肝炎病毒DNA拷贝数,应避免使用含肝素钠和枸缘酸钠的血浆标本,使用不含抗凝剂或含EDTA-K2抗凝剂的无菌试管收集标本。

Objective To explore the effect of different anticoagulants on quantitative assays for HBV DNA using real-time PCR technique. Methods Peripheral blood samples were obtained from 40 patients infected with HBV. Serum and plasma samples from three kinds of anticoagulants such as heparin, trisodium citrate, and dipotassium EDTA were stored at -20℃. Copies of HBV DNA were determined using real - time PCR technique. Paired t-test was used for statistical analysis. Results The logarithmic model to describe the copies of HBV DNA. The logarithmic mean value for copies/mL of HBV DNA in serum was 5.66±2.10, and the value for plasma samples from three kinds of anticoagulants such as heparin, trisodium citrate, and dipotassium EDTA were 5.42± 2.26, 5.36±2.11,5.58±2.16, respectively. Copies of HBV DNA in plasma samples from anticoagulants heparin and trisodium citrate had a significant difference compared with that in control serum samples in this study （P 〈 0.05 ）. The difference was not significant between plasma samples from anticoagulant dipotassium EDTA and the control serum samples （P 〉 0.05）. Conclusion Serum samples should be collected instead of plasma samples from anticoagulants heparin and trisodium citrate when copies of HBV DNA were detected. Plasma samples from anticoag- ulant dipotassium EDTA is also used with quantitative assays for HBV DNA.