Reverse transcriptase-polymerase chain reaction fails to detect peripheral-blood hepatitis C RNA in formalin-fixed liver tissue

Abstract

Currently, one of the major indications for liver transplantation is infection with hepatitis C virus (HCV). Many studies have suggested that recurrent infection with HCV is universal after transplantation. Fastidious techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), have proved to be highly sensitive for detecting HCV RNA in serum and in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) liver tissue. In this study, we wanted to determine whether the identification of HCV RNA in liver tissue by RT-PCR might reflect the detection of circulating HCV RNA in blood within the tissue, rather than implying true tissue infection. We performed RT-PCR for HCV RNA in FFPE liver biopsy specimens taken from 14 donor allografts shortly before and immediately after implantation into recipients. The recipients were known to have HCV RNA in serum and explanted liver tissue, as determined by RT-PCR. We were unable to detect HCV RNA in any of the study samples, either before or after transplantation. In a related study, qualitative and quantitative HCV RNA analyses were performed by RT-PCR and branched DNA (bDNA) amplification, respectively, on serum samples collected pretransplantation and immediately posttransplantation from 10 other patients who underwent transplantation for hepatitis C. HCV RNA was detected in all serum samples before and after transplantation by RT-PCR; however, the bDNA assay detected HCV RNA in only 6 of 10 samples pre-orthotopic liver transplantation (OLT) and in none of the immediately post-OLT samples. In our system, despite the RT-PCR detection of HCV RNA in serum before and after the transplantation, HCV RNA is not detectable in the peripheral blood that accompanies formalin-fixed liver tissue. This implies that RT-PCR detection of HCV RNA in tissue reflects true liver infection, rather than contamination by HCV RNA in accompanying peripheral blood.

Early Detection of Hepatitis C Allograft Reinfection after Orthotopic Liver Transplantation: A Molecular and Histologic Study

“…Our inability to detect HCV RNA by RT-PCR in these specimens indicates that, at least in our system, "background" peripheral blood in liver biopsy specimens does not yield false positivity that theoretically could have been caused by "passenger" HCV RNA in serum or peripheral blood mononuclear cells that accompany the tissue. We have previously shown that this technique in similarly fixed (overnight formalin) tissue from the explanted liver specimens of these patients showed 100% sensitivity and yielded no false positives (13,14). The above results are important in validating the use of RT-PCR to detect HCV RNA in tissue, particularly given the technical difficulties to date using immunoperoxidase (29,36,37) and in situ hybridization techniques (37)(38)(39).…”

“…Another observation that raises the possibility of tissue RT-PCR detection of circulating HCV RNA has been the discovery of HCV RNA in liver biopsy tissue as early as 7 to 12 days post-OLT (5,22). In a preliminary study, however, we failed to detect HCV RNA by RT-PCR in liver biopsies taken from HCV-positive individuals immediately posttransplant (14). Our inability to detect HCV RNA by RT-PCR in these specimens indicates that, at least in our system, "background" peripheral blood in liver biopsy specimens does not yield false positivity that theoretically could have been caused by "passenger" HCV RNA in serum or peripheral blood mononuclear cells that accompany the tissue.…”

“…From this point on, the preparation for both types of specimens remained the same. The extraction was performed using the same procedure described in our previous articles (13,14).…”

“…HCV RNA was amplified using the same combined RT-PCR method described previously (13,14). The primers used target the high conserved 5Ј untranslated region of the HCV.…”

Utilization of Acidophil Bodies in the Diagnosis of Recurrent Hepatitis C Infection after Orthotopic Liver Transplantation

“…The emphasis is therefore on accurate diagnosis of recurrent hepatitis C (RHCV), and its distinction from other causes of elevated liver enzymes or disordered liver function. Reinfection of the allograft as detected by the presence of hepatitis C RNA by RT-PCR is universal, and can occur within weeks of transplantation (15)(16)(17)(18). This however does not necessarily imply hepatitis, so that liver biopsy remains the only available tool to distinguish reinfection from recurrent hepatitis.…”