Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from regular and fine-needle biopsies
Introduction
Infection by HBV affects millions of people worldwide (McMahon, 2005). Replication of HBV virus occurs almost exclusively in hepatocytes, although other cells types may be infected (Umeda et al., 2005). The liver is, therefore, the most relevant site for monitoring the course of HBV infection and antiviral responses. In current clinical practice, HBV infection is monitored primarily by examining serum. In serum, viral DNA, soluble hepatitis B e antigen (HBeAg) or virion-associated viral proteins (e.g. HBsAg and HBcAg) can be detected readily. However, it is still controversial whether the serum viral load always reflects accurately the level and progression of infection in the liver (Iloeje et al., 2006, van der Eijk et al., 2006). A good example is recurrence of HBV infection after apparent clearance of the virus as a result of antiviral therapy (Ito et al., 2004). Indeed, recent studies found that detection of HBV DNA and replication-intermediate cccDNA in the liver, rather than in serum, are better surrogate markers of a true sustained virological response (Alberti, 2003, Werle-Lapostolle et al., 2004, Sung et al., 2005). Clearly, this indicates that when HBV DNA is not detectable in serum, virus can still persist in the liver and can restart replicating once antiviral therapy is stopped. Determination of predictive factor(s) of response to therapy is needed (Zoulim, 2006). Therefore, to be able to assess successfully a sustained virological response in patients on antiviral therapy, the liver is the most relevant site for monitoring the clearance of the virus.
Intrahepatic monitoring of HBV infection can be undertaken using liver tissue biopsies. Detection of intrahepatic viral DNA or RNA has been reported. Although PCR techniques are very sensitive, these do not provide information on the number of infected hepatocytes or the intrahepatic immune response against these cells, as can be observed by immunohistochemistry. Detection of viral antigens, in particular HBsAg and HBcAg, by immunohistochemistry is common practice (Bianchi and Gudat, 1979). However, standardization of these assays is difficult. Furthermore, the use of regular tissue biopsy needles is invasive and could lead to serious complications (Reuben, 2003). As a less traumatic alternative, fine-needle aspiration biopsy has been introduced successfully for the detection of allograft rejection (Hayry et al., 1981, Hayry and Lautenschlager, 1992). The diameter of this needle is significantly smaller (25-Gauge) than the core-needle and therefore fine-needle aspiration biopsy does not require anesthesia and has minimal risk of complication due to damage of intrahepatic blood vessels or biliary tracts. Aspiration biopsy was shown to be as effective for the cytological diagnosis of liver graft rejection as core-needle biopsy (Lautenschlager et al., 1988, Kwekkeboom et al., 2003). Though clearly useful for cytological, immunocytochemical (Kuijf et al., 2002) and flowcytometric analysis (Sprengers et al., 2005), to date the utility of aspiration biopsy for detection of intrahepatic viral antigens has not been reported. As shown previously, aspiration biopsy was shown to be suitable for intrahepatic monitoring of virus-specific T-cell response in patients with chronic HBV (Sprengers et al., 2005).
In the present study, we have investigated the use of flow cytometry for quantitation of viral antigens in regular and aspiration liver biopsy samples from chronic HBV patients. The feasibility of intracellular detection of HBsAg and HBcAg by flow cytometry was demonstrated. Using this method, both the percentages of infected hepatocytes and the relative expression level of viral antigens within the infected cells could be determined.
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Patients
Patients with chronic HBV under surveillance at the Erasmus MC outpatient clinic were included. Patients underwent a liver biopsy as part of their diagnostic evaluation. Control liver biopsies were obtained from patients with chronic HCV or transplant donor livers. The characteristics of patients are summarized in Table 1.
Regular- and fine-needle liver biopsy
Regular biopsy was performed after an ultrasound examination with tru-cut automate core-needle (14 Ga, MD Technologies, Inc., Gainsville, FL, Ref. 763114160X) under standard
Validation of the flowcytometric HBsAg and HBcAg detection
In order to determine the specificity and sensitivity of intracellular staining of HBV antigens by flow cytometry the HBV positive hepatoma cell line, HepG2215, and the parental HBV-free HepG2 cells were used. Cells were fixed and permeabilized and stained with antibodies directed against HBsAg and HBcAg. As shown in Fig. 1, specific staining of both HBsAg and HBcAg was seen in the HepG2215 cell line but not in the HepG2 parental cells. Optimal antibody concentrations for anti-HBsAg were 5 μg/ml
Discussion
Intrahepatic monitoring of viral infection and replication can provide relevant information regarding the course of disease and the response to viral therapy. In the present study, the feasibility and value of flow cytometry analysis for the quantitation of viral antigens in liver biopsy samples of patients with chronic HBV was demonstrated. The percentages HBsAg and HBcAg positive hepatocytes obtained by flow cytometry correlated significantly with the results obtained by immunohistochemistry (
Acknowledgements
The authors like to thank Katja Seggeren, Anthonie Groothuismink and Angela Heijens for excellent technical assistance, Dr. Renate van der Molen, Dr. Jaap Kwekkeboom and Dr. Hanneke van Vuuren for helpful discussions and Danielle Wolvers for critically reading the manuscript.
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