This report describes a pilot study evaluating a multiplex gene analysis assay, relevant for complex diseases with limited tissue availability, which proved reliable for the study of tissue samples of fibrogenic chronic liver diseases using FFPE needle biopsy samples. The optimum quantity of liver tissue for best sensitivity and reproducibility was equivalent to two to three needle biopsy sections 8-μm thick for detection of all genes tested. Using this approach, the expression of ten genes, known to be associated with liver fibrosis and/or HSCs activation, were identified as positively correlating with necroinflammatory activity, whereas two HSC housekeeping genes were identified as negatively correlating with activity, though we could not find a strong correlation between gene expression and fibrosis stage.
We used the bDNA technology QuantiGene™ Reagent System for quantifying mRNA expression. The technical advantage of the sandwich nucleic acid hybridization assay  lies in amplification of reporter signals without enzymatic amplification of the target mRNA, which allows direct measurement of mRNA in tissue homogenates, overcoming issues associated with mRNA extraction. This technology was chosen over a qPCR-based assay method for two primary reasons: 1) QuantiGene™ detection can accurately measure mRNA in FFPE samples whereas qPCR cannot reliably measure mRNA, which has an average size of 100 bases; and 2) QuantiGene™ can be multiplexed more easily than qPCR which is more suited for expression analysis of mRNA extracted from fresh or frozen samples. Prior attempts to employ qPCR on a subset of selected genes in paraffin resulted in no detectable gene expression in paraffin liver RNA and resulted in no detectable gene expression or gene expression beyond the limit of detection (Staten, data not published). Thus, we were not able to use qPCR to compare gene expression between samples or correlate between qPCR and bDNA technologies.
The selection of the genes to be evaluated was made arbitrarily on the basis of a mixed combination of data obtained from human fibrotic liver, a rat BDL model and in vitro culture of immortalized hepatic stellate cells, namely L190 and LX2, and therefore the selected genes are likely related to established inflammation and fibrosis. Other genes could be used in this multiplex assay to address complementary, but different, questions. For example we did not select all of the candidate genes strictly on the basis of a subtraction analysis done by comparing gene expression in fibrotic liver (or activated HSC) and normal liver (or quiescent stellate cells), as described by others previously [9, 10]. This alternative approach may be more appropriate to study the difference of gene expression between disease state and normal liver. However, our approach and the choice of genes used in the present study are more appropriate for investigating the modulation of inflammation and fibrosis between different samples of diseased liver, and could also be applied to verify the modulation of these genes following a period of treatment (for example, predictor of antifibrotic and anti-inflammatory response).
Prognostic factors of rapid progression of liver fibrosis to cirrhosis based on liver biopsy, but also on somatic genetic markers, have been described [11, 12]. For example, Asselah and colleagues  identified 11 genes that differentiate mild from moderate fibrosis based on their respective expression levels. But only two of these genes (COL1A1, TIMP1) overlapped with our study. For identification of potential therapeutic targets, this multiplex assay can be customized with other genes of interest. Liver fibrosis is a disease characterized by collagen deposition  and we included in the multiplex assay multiple genes coding for proteins involved in extra cellular matrix formation, deposition and turnover (COL1A1, COL3A1, COL4A1, ACTA2 and TIMP1); and one gene encoding a component of the cytoskeleton (KRT18). However, in our report, the highest correlation with inflammatory activity stage was observed for annexin 2 (ANXA2). ANXA2 has many described functions but its role in the hepato-biliary system is not fully understood. It is expressed in many cells including hepatocytes, HSCs  and cholangiocytes . ANXA2 is involved in the plasminogen/plasmin system with fibrinolytic effects promoting clot dissolution in alcoholic liver cirrhosis . In inflammatory cholangiopathy ANXA2 can mediate a compensatory effect for the impaired bicarbonate secretion of cholangiocytes through modulation of anion exchanger . ANXA2 has been shown to be expressed as a vitamin D binding protein on hepatocytes and HSCs. Some of these interactions could be important for liver fibrogenesis and inflammation through a TGFβ-independent mechanism .