Polymerase chain reaction analysis of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver biopsies from alcoholics using a simplified and standardized amplification protocol.
Sixty-seven formalin-fixed and paraffin-embedded liver biopsies from HBsAg-negative alcoholics without previous blood transfusions or intravenous drug abuse were analyzed for the presence of low-level hepatitis B virus DNA by the polymerase chain reaction. To simplify and standardize the amplification procedure, aliquots of a complete polymerase chain reaction mix were prepared and frozen for storage; random samples were tested prior to analysis of clinical material. Freezing and storage of the aliquots did not affect the activity of Taq polymerase. One large batch of ready-to-use aliquots could thus be used as a standardized polymerase chain reaction kit for all experiments. The suitability of the extracted material for polymerase chain reaction analysis was tested in two ways. First, the absence of nonspecific polymerase chain reaction inhibitors was demonstrated in all samples by amplifying cloned hepatitis B virus DNA in the presence of extracted material. Second, the integrity of the extracted DNA was tested by amplifying a segment of the beta-globin gene. Twenty-three samples were beta-globin DNA positive and thus contained sufficient amounts of nondegraded DNA. These results emphasize the importance of testing both the absence of nonspecific inhibitors and DNA integrity in DNA samples extracted from fixed tissue. Among the 23 beta-globin positive samples, 12 had cirrhosis (52.1%). Two of these samples were hepatitis B virus DNA positive (8.7%); one of these cases had cirrhosis. Thus, even in the absence of common risk factors, the incidence of hepatitis B virus in this alcoholic population was increased compared to the general population.
von Weizsäcker
F
F
Molecular Hepatology Laboratory, MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA.
Blum
H E
HE
Wands
J R
JR
D000437:Alcoholism / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D001483:Base Sequence; D001706:Biopsy; D004279:DNA, Viral / Q000032:analysis*; D005404:Fixatives; D005557:Formaldehyde; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction
D004279:DNA, Viral; D005404:Fixatives; D015335:Molecular Probes; D005557:Formaldehyde