Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové, Czech Republic.
Department of Preventive Medicine, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic.
Folia Biol (Praha). 2022;68(5-6):201-205. doi: 10.14712/fb2022068050201.
We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalatetreated samples: 18.5 and 18.9, respectively. Significantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.
我们比较了实时 PCR 分析在不同抗凝剂（EDTA、肝素、氟化钠和草酸钾）处理的静脉血样本或凝固的静脉血样本中 FII（c.*97G>A、G20210A）和 FV Leiden（c.1601G>A）易栓突变的效率；对 100 例野生型个体的样本进行了测试。对经过 90°C 加热的基因组 DNA 提取物和全血标本进行实时 PCR 分析；随后评估循环阈值（Ct）值。对 EDTA 血样 DNA 提取物中的 FII 基因检测进行实时 PCR 分析，结果显示中位 Ct 值为 19.3。肝素化血液和氟化钠与草酸钾处理的样本中，DNA 提取物的 Ct 值相似：分别为 18.5 和 18.9。凝固血提取物的 Ct 值明显较高，中位数分别为 20.6（含惰性分离胶的管）和 20.5（不含凝胶的管，均 P<0.001）。FV 实时 PCR 分析的数据与 FII 检测非常相似。在改良的全血中，与 EDTA、氟化钠与草酸钾以及凝固血样本相比，肝素盐处理的样本在两种检测中均显示出显著更低的 Ct 值（P<0.001）。我们的结果表明，实时 PCR 分析易栓突变不受收集管中肝素盐的存在的负面影响。当需要进行易栓突变的 DNA 分析时，不同抗凝剂的血液样本可以相互替代。
