临床常见干扰因素对ctDNA EGFR T790M突变检测的影响

目的探讨临床常见干扰因素对血浆循环肿瘤DNA(ctDNA)表皮生长因子受体(EGFR)T790M突变检测的影响。方法收集7例确认T790M为野生型的正常献血者血浆,添加不同浓度T790M突变质粒及单一干扰物(三酰甘油、胆红素或血红蛋白)模拟干扰血浆,提取DNA后,采用超级突变扩增阻滞系统(SuperARMS)进行T790M突变检测,每份样本重复检测3次,记录DNA浓度、DNA纯度、Super-ARMS检测内控Ct值及T790M突变ΔCt值,应用SPSS 22.0软件对检测结果进行统计学分析。结果临界阳性突变样本DNA浓度极易受胆红素(≥85.5μmol/L)及三酰甘油(≥9.25 mmol/L)干扰(P<0.05),但其纯度不易受干扰;强阳性突变样本DNA浓度可受低浓度胆红素(85.5μmol/L)及高浓度血红蛋白(2.0 g/L)干扰(P<0.05),且纯度受三酰甘油(≥9.25 mmol/L)和血红蛋白(0.5~1.5 g/L)干扰(P<0.05)。脂血对Super-ARMS检测T790M突变的影响最显著,临界阳性、强阳性突变样本内控Ct值及T790M突变ΔCt值均可受三酰甘油(≥9.25 mmol/L)干扰(P<0.05);此外,强阳性突变样本内控Ct值受较高浓度胆红素(≥256.5μmol/L)干扰,T790M突变ΔCt值可受中等浓度血红蛋白(≥1.0 g/L)干扰(P<0.05)。结论不同浓度的血红蛋白、三酰甘油和胆红素对SuperARMS检测ct DNA EGFR T790M突变的各项参数可产生不同程度的影响,在临床实际检测中应尽量避免使用含干扰因素的血浆。

Influence of common clinical interference factors on the detection of ctDNA EGFR T790M mutation

Objective To study the influence of common clinical interference factors on the detection of epidermal growth factor receptor(EGFR)T790M mutation of circulating tumor DNA(ctDNA)in plasma.Methods T790M wild-type plasma from 7 healthy donors was collected.Different concentrations of T790M mutant plasmid and certain interference factor(triglyceride,bilirubin and hemoglobin)were added into the T790M wildtype plasma to simulate interfered plasma.T790M mutation was detected by super amplification refractory mutation system(Super-ARMS)after DNA extraction.Each sample was detected for 3 times,the concentration and purity of DNA,Ct values of internal control andΔCt values of T790M mutation were recorded.SPSS 22.0 software was used for statistical analysis.Results While the concentration of DNA of critically positive mutant samples could be affected by bilirubin(≥85.5μmol/L)and triglyceride(≥9.25 mmol/L)(P<0.05),the purity of DNA of them could not be affected.As low concentration of bilirubin(85.5μmol/L)and high concentration of hemoglobin(2.0 g/L)affected the concentration of DNA of strongly positive mutant samples(P<0.05),triglyceride(≥9.25 mmol/L)and hemoglobin(0.5-1.5 g/L)also affected the purity of DNA(P<0.05).Lipemia had influence on the detection of T790M mutation by Super-ARMS,and triglyceride(≥9.25 mmol/L)affected the Ct values of internal control andΔCt values of T790M mutation of both strongly and critically positive mutant samples(P<0.05).Otherwise,high concentrations of bilirubin(≥256.5μmol/L)affected the Ct values of internal control of strongly positive mutant samples,and middle concentrations of hemoglobin(≥1.0 g/L)affected theirΔCt values of T790M mutation(P<0.05).Conclusions Different concentrations of hemoglobin,triglyceride and bilirubin have different effects on several indicators of detecting ctDNA EGFR T790M mutation by Super-ARMS.Plasma containing interference factors should be avoided in practical clinical detection as far as possible.