Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients.
BACKGROUND
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up.


OBJECTIVE
To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Takkenberg
R Bart
RB
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. r.b.takkenberg@amc.nl
Zaaijer
Hans L
HL
Menting
Sandra
S
Weegink
Christine J
CJ
Terpstra
Valeska
V
Cornelissen
Marion
M
Dijkgraaf
Marcel G W
MG
Jansen
Peter L M
PL
Reesink
Hendrik W
HW
Beld
Marcel G H M
MG
D000328:Adult; D015925:Cryopreservation; D004270:DNA, Circular / Q000032:analysis* / Q000097:blood; D004279:DNA, Viral / Q000032:analysis*; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000188:drug therapy* / Q000821:virology*; D006801:Humans; D008099:Liver / Q000821:virology*; D016612:Paraffin Embedding
D004270:DNA, Circular; D004279:DNA, Viral; D006513:Hepatitis B e Antigens