论文部分内容阅读
目的 :慢性乙型肝炎患者外周血单个核淋巴细胞 (Peipheralbloodmononuclearcells,PBMC)不同洗涤方法对HBVDNA检测的影响。方法 :淋巴细胞分离液常规密度梯度法分离 1例血清HBVDNA阳性慢性乙型肝炎患者PBMC后 ,分别采用常规洗涤法和改良洗涤法 (后者即在常规洗涤法的基础上第 2、 4次洗涤前不重悬细胞 ,直接加入洗液后离心 ) ,计算细胞收获得率和细胞存活率 ,荧光定量PCR法检测 1、 3、 5次洗液样本和细胞样本的HBVDNA ,重复实验 3次。结果 :常规法第 1、 3、 5次洗液样本HBVDNA均阳性 ,改良法洗涤 5次后洗液样本HBVDNA均阴性 ;改良法洗涤的细胞得率和细胞存活率分别为 95 %和 98% ,常规法洗涤的细胞得率和细胞存活率分别为 71%和 83% ;不论常规洗涤法和改良洗涤法收获的PBMC均可检出HBVDNA。结论 :慢性乙型肝炎患者PBMC改良洗涤法优于常规洗涤法 ,适用于PBMC中HBVDNA荧光定量PCR检测。
Objective: To investigate the effect of different washing methods on the detection of HBVDNA in peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis B (CHB). Methods: PBMCs from one patient with serum HBVDNA positive chronic hepatitis B were separated by routine density gradient method. The routine washing method and modified washing method were used (the latter was the second and the fourth washing based on the conventional washing method Before resuspending the cells, directly adding the lotion and centrifuging), calculating the cell harvesting rate and the cell survival rate, detecting the HBV DNA in one, three or five washings and cell samples by the fluorescence quantitative PCR method, and repeating the experiment three times. Results: The HBVDNA of the first, third, and fifth washings were all positive by conventional method. The HBVDNA of the wash samples was negative after five times of washing by modified method. The cell yield and cell survival rate of the washing method were 95% and 98% The cell yield and cell survival rate of the conventional method were 71% and 83%, respectively; HBVDNA could be detected in the PBMC harvested both by the conventional washing method and the modified washing method. Conclusion: The improved washing method of PBMC in patients with chronic hepatitis B is superior to the conventional washing method, which is suitable for the detection of HBV DNA in PBMC by fluorescence quantitative PCR.