Institute of Clinical Biochemistry and Diagnostics, Charles University Hospital, Hradec Králové, Czech Republic.
Physiol Res. 2022 Jul 29;71(3):439-445. doi: 10.33549/physiolres.934883. Epub 2022 May 26.
High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.
COVID-19 患者的血栓形成和静脉血栓栓塞发生率较高。在这项研究中，我们专注于分析未经标准 DNA 提取步骤进行的血栓形成倾向突变。从 COVID-19 阳性门诊患者的 100 份 DNA 提取物和改良全血样本中进行了 FV 基因 Leiden 突变和 FII 基因 G20210A 突变的实时 PCR，并评估了它们的循环阈值 (Ct) 值。在提取物中，健康的纯合子 (wt/wt)、杂合子 (M/wt) 和 Leiden 突变的纯合子携带者 (M/M) 提供了 18.5、19.4/22.0 和 20.9 的中位 Ct 值。在全血中，Ct 值为 25.3 (wt/wt)、24.8/27.2 (M/wt) 和 26.9 (M/M)。提取物中 G20210A 的中位 Ct 值为纯合子 (wt/wt) 的 19.6，杂合子携带者的 19.7/20.4。全血样本中 G20210A 突变的健康纯合子的 Ct 值为 23.9，杂合子的 Ct 值为 26.3/27.2。未发现 G20210A 的纯合子受试者，也未发现 Leiden 和 G20210A 突变的双重杂合子。尽管 Ct 值存在显著差异，但基因分型显示 DNA 提取物和全血样本的结果完全一致。在 28 天的深冻过程中，全血样本中的 DNA 分子的完整性和扩增能力没有明显变化，期间进行了四次解冻循环。总之，我们展示了一种通过对 COVID-19 阳性患者的改良全血进行实时 PCR 检测血栓形成倾向突变的新方案。血液改良可靠、简单、廉价，节省了整个实验室过程的成本和周转时间。
