Department of Gastroenterology and Hepatology, AMC Liver Center, Center for Infectious Disease and Immunology (CINIMA), University of Amsterdam and Royal Tropical Institute, KIT biomedical Research, Amsterdam, The Netherlands.
Eur J Gastroenterol Hepatol. 2010 Aug;22(8):952-60. doi: 10.1097/MEG.0b013e3283376a63.
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up.
To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue.
Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used.
The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P<0.05). In HBeAg positive patients, no difference in intrahepatic cccDNA levels were seen between patients with active (histological activity index score>3; HBV DNA>20 000 IU/ml) and inactive hepatitis (histological activity index score</=3). In HBeAg negative chronic hepatitis B patients, intrahepatic cccDNA levels were significantly higher in patients with active hepatitis (P=0.004 and 0.001).
Recovery of hepatocytes and cccDNA in FFPE tissue was lower, but intrahepatic cccDNA in FFPE biopsies were comparable with cryo-preserved liver tissue. Therefore, FFPE liver tissue is an attractive alternative for cccDNA analysis when cryo-preserved tissue is not available.
乙型肝炎病毒（HBV）共价闭合环状 DNA（cccDNA）可能成为治疗结果或长期随访的重要预测指标。
检测福尔马林固定、石蜡包埋（FFPE）组织中的 cccDNA，并与冷冻保存的肝组织进行比较。
收集了 56 例慢性乙型肝炎患者的肝活检组织。其中 37 例患者的肝活检组织同时有冷冻保存和 FFPE 组织。用 1ml 二甲苯提取石蜡，然后用 100%乙醇和丙酮。用选择性引物检测 cccDNA，用商品化 Taqman β-肌动蛋白对照试剂盒检测肝细胞的定量。
80%的 FFPE 组织和 100%的冷冻保存组织中均检测到 cccDNA。FFPE 肝组织中肝细胞和 cccDNA 的回收率约低 100 倍，但肝内 cccDNA 水平相当。在 FFPE 和冷冻保存的肝组织中，肝内 cccDNA 水平与 HBV DNA、乙型肝炎 e 抗原（HbeAg）和血浆 cccDNA 水平密切相关。HbeAg 阳性慢性乙型肝炎患者的肝内 cccDNA 水平明显高于 HBeAg 阴性患者（P<0.05）。在 HBeAg 阳性患者中，活动性（组织学活动指数评分>3；HBV DNA>20000 IU/ml）和非活动性肝炎（组织学活动指数评分<=3）患者的肝内 cccDNA 水平无差异。在 HBeAg 阴性慢性乙型肝炎患者中，活动性肝炎患者的肝内 cccDNA 水平明显较高（P=0.004 和 0.001）。
FFPE 组织中肝细胞和 cccDNA 的回收率较低，但 FFPE 活检组织中的肝内 cccDNA 与冷冻保存的肝组织相当。因此，当无法获得冷冻保存的组织时，FFPE 肝组织是分析 cccDNA 的一种有吸引力的替代方法。
