| BackgroundInfection with hepatitis B virus (HBV) is a major global health problem. HBV was documented to be the main cause of severe, acute, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Especially, China is a dominant country with HBV infection. A recent study reported that HCC caused 384,000 deaths per year in China. In particular, severe hepatitis is a devastating liver disease with a very high mortality of 80%, as the condition progresses, it can lead to other complications. Therefore, it is of extremely important significance how to identify and monitor various kinds of type B hepatitis and hepatic fibrosis and early HCC.As is known to all, liver cells damage of hepatitis B patients is a common phenomenon and the degree of liver cells injury is different in various kinds of liver diseases. There has remained controversial that the present laboratory specific parameter (such as serum ALT,HBV DNA et al) could be applied in evaluating liver cells injury degree. AFP is a very valuable index in identifying and diagnosing HCC, but there is a certain false positive and false negative.Plasma DNA is a cell-free extracellular DNA, and it is a form of circulating DNA. The high level of circulating plasma DNA is not only present in cancer; but also it is associated with infectious diseases, trauma, inflammation and pregnancy. Because liver cells injury degree is different in various kinds of liver diseases under the condition of tumor or infection or in?ammatory, and the amount of circulating DNA releasing from liver cells is different. So it is possible to assess liver cells injury degree with hepatitis B patients and identifying HCC by measuring plasma DNA. Up to present, most of researches pay more attention to explore the relationship between circulating DNA and HCC with HCV infection, whereas little of literature with regard to the association between circulating plasma DNA and hepatitis B diseases and HCC with HBV infection have been reported.ObjectiveThe aim of this study is to quantify plasma DNA in liver disease patients with HBV infection by using duplex real-time quantitative PCR assay that was designed by oneself, and to accurately assess the degree of liver cells injury for patients with hepatitis B and its clinical application value in identifying and diagnosing all kinds of liver diseases, and to evaluate its diagnosis efficacy comparing with the present laboratory conventional indicators.MethodsPeripheral blood samples were collected from a cohort of 185 patients with hepatitis B which are divided into four groups, severe hepatitis with 30 cases, acute hepatitis with 20 cases, chronic B hepatitis with 90 cases, and liver cirrhosis with 45 cases. 200 healthy controls were enrolled. Circulating DNA was extracted from plasma by means of the magnetic bead extraction assay and quantified with novel duplex real-time quantitative PCR assay, respectively. The degree of liver cell injury for patients with severe hepatitis was accurately assessed.Peripheral blood samples were collected from 152 patients with liver diseases which are divided, into three groups, HCC with 59 cases, chronic B hepatitis with 56 cases, and liver cirrhosis with 37 cases. 200 healthy controls were enrolled. Circulating DNA was extracted from plasma with the magnetic bead extraction assay and quantified with the duplex real-time quantitative PCR assay, respectively. We evaluated clinical application value in identifying and diagnosing HCC. Results1,Comparison between severe hepatitis and other hepatitis BPlasma DNA levels of hepatitis B patients were significantly higher than that of healthy controls (104.2 vs 18.7 ng/ml (median), P = 0.0000). The significant difference in plasma DNA concentration was found between severe hepatitis and acute hepatitis (P = 0.0018), and chronic B hepatitis (P = 0.0000), and liver cirrhosis (P = 0.0000); On the other hand, the median values of serum ALT from hepatitis B patients were 107.5 U/L, much higher with respect to the healthy controls (24.1 U/L, P = 0.0000). Levels of serum ALT were significantly different between acute hepatitis and severe hepatitis (P = 0.0024), while there was no remarkable difference between severe hepatitis and chronic B hepatitis (P = 0.0600), and liver cirrhosis (P = 1.0000). Moreover, in distinguishing severe hepatitis from liver cirrhosis and chronic B hepatitis, the power of plasma DNA assay was notably superior to that of ALT by the receive operating characteristic (ROC) curves (AUC, 0.95 vs 0.51, P = 0.0000; 0.86 vs 0.34, P = 0.0000).In addition, Serum bilirubin median levels of severe hepatitis (195.5μmol/ L), more than 10 times the normal upper limit, are the highest among hepatitis B diseases. Meanwhile, median levels of serum HBV DNA from hepatitis B patients (1.1×106 copies/ml) also rise significantly, but no remarkably difference was noted among four groups of hepatitis B (P = 0.7378). By the Spearman's rank correlation analysis, plasma DNA levels are positively associated with the liver cell injury degree (r = 0.62), but ALT level is not related to that (r =0.11). There is no remarkably association between plasma DNA and serum ALT (r = 0.27).2,Comparison between HCC and other hepatitis BPlasma DNA levels of liver disease patients were significantly higher than that of healthy controls (105.5 vs 18.7 ng/ml (median), P = 0.0000). Plasma DNA levels were 260.2 (96.9~435.6),90.6 (60.7~134.2) and 74.8 (44.1~127.3) ng/ml in HCC,chronic B hepatitis and liver cirrhosis, respectively. The significant difference in plasma DNA concentration was found between HCC and chronic B hepatitis (P = 0.0000), and liver cirrhosis (P = 0.0000); the median values of serum AFP from liver disease patients were 16.2 ng/ml, much higher with respect to the healthy controls (9.6 ng/ml, P = 0.0000). Levels of serum AFP were significantly different between HCC and chronic B hepatitis (P = 0.0033), and liver cirrhosis (P = 0.0033). The significant difference in plasma DNA level was found between HCC with HBsAg positive and with HBsAg negative (310.9 vs 106.7 ng/ml (median), P = 0.0290), while there was no remarkable difference in serum AFP level between the two groups (124.0 vs. 66.5 ng/ml (median), P = 0.2090). Moreover, in distinguishing HCC with HBsAg positive from liver cirrhosis and chronic B hepatitis, the power of plasma DNA assay was superior to that of srum AFP by the ROC curves (AUC, 0.82 vs 0.72; 0.77 vs 0.70). By the Spearman's rank correlation analysis, there is no significantly correlation between plasma DNA and serum AFP (r = 0.37).ConclusionThis is the first systemic study regarding the measurement of plasma DNA in all kinds of liver disease patients by using the duplex real-time quantitative PCR assay with internal control. The results show that plasma DNA may be considered as a robust predictive marker in accurately evaluating liver cell injury degree of severe hepatitis patients and its clinical application value in identifying HCC patients combining with other laboratory indexes. |
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