Technical Notes No Inhibitory Effect of Heparinized Blood on Real-Time PCR Analysis of Thrombophilic Mutations

Abstract

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 anti­coagulant 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 oxalate-treated 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.