摘要研究甲状腺细针穿刺标本行BRAF基因检测过程中常见的问题,探讨需要重复实验样本的条件,并提出一套更为完善的检测及判读过程.方法 收集中日友好医院2012年2月至2018年7月期间送检的8 644例甲状腺细针穿刺标本,使用即时定量PCR法检测BRAF基因突变.对实验结果模棱两可或与临床或细胞学诊断不符的237例样品进行重复实验.结果 最终BRAF基因突变阳性率22.0%(1 897/8 625),DNA质控不合格样品19例.样品的平均内质控荧光Ct值为16.061,阳性标本的平均荧光值为19.147.237例重复实验中,第一次DNA质控不合格重复后仍然不合格者占51.4%(19/37),合格者占48.6%(18/37),其中阳性1例,阴性17例.阴性标本重复40例,结果均未发生改变.第一次实验阳性,BRAF基因与内质控基因荧光Ct值差值在8~12之间的样品,重复后40.4%(40/99)的标本转变为阴性;差值在12以上的标本,重复后69.8%(37/53)的样品转变为阴性.BRAF突变的灵敏度与特异性分别为83.97%与96.94%.结论 推荐将BRAF荧光Ct值与内质控荧光Ct值的差值作为判读实验结果是否可靠的一个指标,一般差值大于8的样品应当进行重复实验.

abstractsstudy common problems in BRAF gene mutation detection, and conditions for repetition testing using thyroid fine needle aspiration specimens. Methods A total of 8 644 cases of thyroid fine?needle aspiration specimens at China?Japan Friendship Hospital were collected between February, 2012 and July, 2018. BRAF gene mutation was detected by real?time PCR. Repeat testing was performed in 237 cases when the results were inconsistent with clinical or cytological diagnosis or when uncertain results were obtained. Results The final positive rates of BRAF mutation was 22.0% (1 897/8 625). Nineteen cases were excluded due to inadequate DNA samples. The average Ct value of internal quality control was 16.061, and the average Ct value of the positive samples was 19.147. Among 237 repeat tests, 51.4% (19/37) continued to have poor DNA quality and 48.6% (18/37) had adequate DNA resulting in 1 positive case and 17 negative cases. In 40 repetition of initial negative cases, results were unchanged. In initial positive cases, 40.4% (40/99) with a difference of Ct value (between BRAF gene and internal quality control) between 8 to 12 turned negative after repetition, 69.8% (37/53) of these cases with a difference of more than 12 turned negative after repetition. The sensitivity and specificity of BRAF mutation were 83.97% and 96.94%, respectively. Conclusions Difference between BRAF gene Ct value and internal quality control Ct value is recommended as a reliability index for the test result. Cases with a difference greater than 8 should be subjected to repeat testing.