Detection of Hepatitis C by RT-PCR in Formalin-Fixed Paraffin-Embedded Tissue from Liver Transplant Patients

Abstract

The histopathologic alterations of hepatitis C virus (HCV) infection of the liver overlap with those of other diseases, making interpretation of liver biopsy specimens in some cases insufficient to render a diagnosis. Although HCV infection can be confirmed by detection of circulating anti-HCV antibodies, immunocompromised liver transplant recipients are often unable to mount an immunologic response to the virus, resulting in false-negative serologic testing. We describe the comparison of reverse transcription-polymerase chain reaction (RT-PCR) with histopathology, serology, and immunohistochemistry for the diagnosis of HCV. Sixty-three formalin-fixed, paraffin-embedded tissue samples (40 needle biopsy specimens and 23 native liver resection specimens) from 35 transplant patients were analyzed by use of a novel method of RNA extraction followed by nested PCR for HCV as well as albumin mRNA as an internal control. HCV was detected by RT-PCR in 50 of 51 (98%) paraffin sections of liver from transplant patients with circulating anti-HCV antibodies, 15 of which lacked characteristic histologic features of HCV infection. Overall, there were no false-negative results in 36 needle biopsy specimens from patients with hepatitis C infection, but three negative results were seen in end-stage cirrhotic native livers resected from HCV-infected patients. No false-positive test results were seen among 21 negative controls (10 liver samples from immunocompetent patients with abnormalities unrelated to hepatitis C and 11 liver biopsies from immunocompetent patients without histologic evidence of liver disease). In comparison, immunohistochemistry using antibody TORDJI-22 was positive for HCV in only 15 of 32 (47%) needle biopsies positive by RT-PCR. Our results indicate that RT-PCR is a more sensitive and specific method of detecting hepatitis C in routinely processed paraffin sections of formalin-fixed liver biopsy specimens than histopathologic examination or immunohistochemistry.

High levels of hepatitis C virus RNA in native livers correlate with the development of cholestatic hepatitis in liver allografts and a poor outcome

“…20 Another study showed that 8 to 24 hours of formalin fixation leads to loss of RNA by a factor of 100 to 1,000. 21 However, long-term paraffin storage has not been shown to be deleterious in the preservation of HCV RNA. In our study, specimen fixation was consistently less than 8 hours; therefore, we are confident there has been minimal degradation of HCV RNA in the tissues studied.…”

Detection of hepatitis C virus RNA in paraffin‐embedded tissues from patients with non‐Hodgkin's lymphoma

“…There was no evidence of HIV infection in our cases All the samples were reevaluated by an expert pathologist (ME) and classified according to the REAL classification [12]. The Enhanced Avian RT-PCR Kit (Sigma, St. Louis, MO) was used both in tissue and in serum in order to obtain HCV-RNA.…”

Nonspecificity of monoclonal antibody tordji-22 for the detection of hepatitis C virus in liver transplant recipients with cholestatic hepatitis

“…Tordji-22 has recently been used to detect HCV antigens in both nontransplant 19,20 and posttransplantation liver tissue. 20,21 Initially, our results indicated a strong correlation between the location of HCV antigens and areas of liver injury, particularly regions of hepatocyte ballooning. Further testing showed cross-reaction of this monoclonal antibody with host epitopes present in non-HCV liver transplant tissue.…”

“…[19][20][21]30 Therefore, the claim that this monoclonal antibody detected HCV antigen in severely damaged liver tissue from a case of fulminant hepatitis must be viewed with caution. 19 In support of our finding, other groups have reported that the commercial monoclonal antibody Tordji-22 has been associated with both low sensitivity 21,31 and nonspecificity 32 when tested on chronic HCV-infected patients and HCV-positive liver allograft recipients. Vartanian et al 30 observed diffuse, cytoplasmic staining of biopsy specimens from patients with HCV, HBV, and nonviral liver disease tested with Tordji-22.…”