title: Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.
abstract: The polymerase chain reaction (PCR) was used to amplify viral or oncogene sequences from frozen or formalin-fixed, paraffin-embedded tissue sections. Methods for preparing fixed, embedded colonic tissue for PCR amplification of c-K-ras sequences from genomic DNA and for amplification of viral DNA from other tissues, including brain, lung, and liver, were evaluated. The effect of formalin fixation on the efficiency of amplification was also determined. While there seemed to be only a modest variation in the efficiency of the PCR for amplification of single-copy human genes, regardless of the methods used for tissue preparation, amplification of viral DNA sequences against a human genomic DNA background was more efficient when the DNA was purified to some degree before amplification of the tissue. We used the PCR to examine frozen and fixed embedded tissue sections for the presence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA. One patient with a heart-lung transplant succumbed to a lymphoproliferative disorder, and EBV genome was present in tissues with abnormal lymphoid infiltrates. CMV was also present in bronchial lavages from the same patient, where cytologic diagnosis was not apparent. Another patient with a liver transplant showed CMV genome in multiple liver biopsies, with negative histologic results for CMV. In vitro DNA amplification with the PCR demonstrated sensitivity superior to that of histology in detecting CMV and EBV in the cases examined.
authors.affiliation: Department of Pathology, Texas Children's Hospital and Baylor College of Medicine, Houston.
authors.forename: B B
authors.initials: BB
authors.lastname: Rogers
authors.forename: L C
authors.initials: LC
authors.lastname: Alpert
authors.forename: E A
authors.initials: EA
authors.lastname: Hine
authors.forename: G J
authors.initials: GJ
authors.lastname: Buffone
mesh_terms: D000328:Adult; D001344:Autopsy; D001706:Biopsy; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D004064:Digestive System / Q000187:drug effects / Q000378:metabolism; D004068:Digestive System Physiological Phenomena; D005260:Female; D005404:Fixatives; D005557:Formaldehyde / Q000494:pharmacology; D005784:Gene Amplification; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016031:Liver Transplantation; D008232:Lymphoproliferative Disorders / Q000382:microbiology / Q000473:pathology; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras)
chemical_list: D004279:DNA, Viral; D005404:Fixatives; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)